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Full text of "Characterization of the pathogenesis of amelanosis in the Smyth line chicken : a model of the human autoimmune disease vitiligo"

CHARACTERIZATION OF THE PATHOGENESIS OF AMELANOSIS IN THE 
SMYTH LINE CHICKEN: A MODEL OF THE HUMAN AUTOIMMUNE DISEASE 

VITILIGO 



By 

EDMUND C. LEUNG 












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1998 



Copyright 1998 

by 

EDMUND C. LEUNG 















In loving rememberance of my grandmother Gertrude Chur Ho 



in 



ACKNOWLEDGMENTS 



There are many people I want to acknowledge in making this project possible. 
First of all, I am appreciative of my mentor Dr. Wayne McCormack for the opportunity to 
join his lab and for the distinction of being his first graduate student. I thank him for 
passing on his knowledge and experience. 

I thank my committee members Drs. Mark Atkinson, Maureen Goodenow, Ward 
Wakeland, and Thomas Rowe for all their suggestions, support, and encouragement. I 
thank Dr. Goodenow for her gift of the LTR probe in my endogenous virus study. 

For all the work on the live chickens, I want to thank Drs. J. Robert Smyth, Jr., 
and Gisela Erf for assistance in establishing colonies of Smyth and Brown line chickens. 
Without their insight I would not have been able to develop this project. Drs. Jack 
Gaskins, Gary Butcher, Victor Apanius, Ben Mather, and Richard Miles were all 
instrumental in my trials and errors in learning how to perform phlebotomies on chickens 
and to perform injections. I am thankful for the many, many hours provided by almost a 
dozen University of Florida undergraduate students who were willing to come faithfully 
to our poultry facilities even in rainy weather. They learned how to tame these chickens 
enough to move them one at a time from a pen. Without them I could not have performed 
the cell and serum injections, the serum collection, and plucked feathers every two weeks. 



IV 



So I thank Sharon Richertson, Brad Copley, Jaime Sanchez, Nilesh Patel, Randy 
Scarboro, and Shally Wang. May they regard the experience as useful to their careers. 

I thank Bruce Glick for his suggesting that I use cyclophosphamide rather than 
irradiation to immunosuppress the chickens. I wish I had followed this advice. I thank 
Robert E. Boissy for his suggestions as well. 

I thank Karen Achey for all her efforts in sequencing. I thank Pat Glendon for her 
assistance in analyzing proteins. Rose Pratt did so much to provide cryosections of 
regenerating feathers. I am grateful to Paul Kubilis for his advice on statistical analyses. 

And I thank the members of the McCormack lab, Cheryl Spence, Luke Utley, 
Javier Sanchez-Garcia, Alex Aller, Christy Myrick, David Gill, Kim Taylor, Neha Sahni, 
and Claudia Lazo de la Vega, and many of the volunteer students already mentioned for 
their friendship and technical support. 

I thank my family and my friends who have made their own sacrifices to help me 
see this project come to fruition. I especially also want to thank Dr. Jin Xiong She for 
believing in my potential and offering a postdoctorate position to me. 












TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS iv 

LIST OF TABLES ix 

LIST OF FIGURES x 

ABSTRACT xii 

CHAPTERS 

1. INTRODUCTION 1 

Autoimmunity 1 

Tolerance Mechanisms 3 

Loss of Immunological Tolerance 7 

Mechanisms of Autoimmunity 7 

Regulation of Autoimmune Responses 13 

Autoimmune Diseases Cause by Antibodies 14 

Autoimmune Diseases Caused by T cells 16 

Vitiligo in Humans 16 

Melanocyte Biology 17 

Vitiligo Pathology 18 

The Association of Vitiligo with Other Autoimmune Diseases and the 

Genetics of Vitiligo Susceptibility in Humans 20 

The C57BL/6J-viY/wY Mouse Model for Vitiligo 21 

The Smyth Line (SL) Chicken Animal Model for Vitiligo 22 

Melanocyte Biology 26 

Amelanosis Pathology in the Smyth Chicken 30 

Genetics of Vitiligo Susceptibility in SL Chickens 33 

Chicken Immunology 33 

Chicken Immunoglobulin Genes and B Cell Development 33 

Chicken T Cell Receptor Genes and T Cell Development 35 

T Cell Repertoire Analysis 40 

Other Chicken Models of Autoimmunity 42 

Limitations in the Use of the Chicken Animal Model 43 

Rationale for This Study 45 



VI 



2. ADOPTIVE TRANSFER OF AMELANOSIS IN THE SMYTH LINE 
CHICKEN 48 

Introduction 48 

Materials and Methods 55 

Animals 55 

Sex Determination by PCR 56 

Immunosuppression of the Host Animals 57 

Preparation of the SL Donor Cells and Cell Injections 57 

Smyth Line Serum Collection and Preparation 57 

Cell Lines and Source 58 

Immunoblotting 58 

Histology 59 

Results 59 

Observations of the UF Colony of Smyth Line Chickens 59 

Adoptive Cell Transfer Experiments 64 

Serum Transfer Experiment 72 

Western Blot Analysis 76 

Discussion 79 

3. T CELL RECEPTOR y REPERTOIRE ANALYSIS OF THE EXPANDED 
PERIPHERAL BLOOD y8 T CELL POPULATION DURING AVIAN 
VITILIGO 85 

Introduction 85 

Materials and Methods 89 

Animals 89 

RT-PCR and Cloning 90 

DNA Sequence Comparisons 91 

Results 91 

Phenotype of Birds Used for Repertoire Analysis 91 

TCR Vy 6 Repertoire Analysis 92 

CDR3 Length and Amino Acid Composition 100 

Jy Usage 100 

Discussion 100 

4. ENDOGENOUS VIRAL LOCI IN THE SMYTH LINE CHICKEN: A 

MODEL FOR THE AUTOIMMUNE DISEASE VITILIGO 106 

Introduction 106 

Materials and Methods 112 

Southern Blot Analysis 112 

Statistical Analyses 113 

Results 113 

Phenotypic Analysis of SL Sample Population 113 



vn 



Southern Blot Analysis of BL and SL ev Loci 114 

Comparison of BL and SL ev Genotypes 122 

Comparison of SL Progressor and SL Nonprogressor ev 

Genotypes 124 

Discussion 126 

5. SUMMARY AND FUTURE DIRECTIONS 132 

LIST OF REFERENCES 139 

BIOGRAPHICAL SKETCH 159 



vni 



LIST OF TABLES 

Table page 

2-1. Amelanosis incidence in the UF Smyth line colony 60 

2-2. Adoptive transfer of amelanosis with single transfers of SL lymphocytes 66 

2-3. Progression of amelanosis in 5 BL5 hosts after adoptive transfers of SL 

lymphocytes 67 

2-4. Adoptive transfer of amelanosis with multiple transfers of SL lymphocytes ... 71 

2-5. Bio Rad protein assay of gamma globulin pools and selected 

serum samples 74 

2-6. Adoptive transfer of SL gamma globulins into 6 week old BL10 hosts 75 

3-1 . Amelanosis stage of Smyth Line chickens at ages 2-25 weeks 91 

4-1. Smyth line (SL) chicken phenotypes 115 

4-2. Frequencies of ev loci detected in BL and SL chickens 118 

4-3. Frequencies of ev loci detected in SL progressing (p) and nonprogressing 

(np) chickens 125 



IX 



LIST OF FIGURES 

Figure page 

1-1. A female Smyth line chicken displaying amelanosis of stage 4 23 

1-2. A group of Smyth line chickens at various stages of amelanosis 23 

1-3. A typical pair of parental Brown line chickens 24 

1-4. Model of developing feather showing the arrangement of barb ridges 27 

1-5. A cross section of a feather shaft and barb 28 

1-6. A single barb ridge 29 

1-7. Chick thymocyte development 36 

1-8. Models depicting the V, D, J, and C gene segments of the T cell receptors .... 39 

2-1. Frequencies of Smyth line females 61 

2-2. Frequencies of Smyth Line males 62 

2-3. Frequencies of Smyth line females and males 63 

2-4. BL5-1 1 1 , a Brown line adoptive transfer host displaying stage 3 amelanosis . . 69 

2-5. BL5-1 15, a Brown line adoptive transfer host displaying stage 2 amelanosis . . 69 

2-6. Adoptive cell transfer hosts show antimelanocyte antibody profile typical 

of SL chickens 77 

3-1. Partial nucleotide sequences of rearranged TCR Vy 1 genes 94 

3-2. Partial nucleotide sequences of rearranged TCR Vy 2 genes 96 

3-3. Partial nucleotide sequences of rearranged TCR Vy 3 genes 98 

x 



3-4. Predicted amino acid sequences of rearranged TCR Vy genes 101 

4-1 . Southern blot analysis of ev loci detected as BamHl restriction fragments .... 116 

4-2. Southern blot analysis of ev loci detected as EcoKl restriction fragments .... 1 17 

4-3. BL and SL chickens have similar total numbers of ev loci 121 

4-4. SL chickens have more ev-SL loci than BL chickens 123 









XI 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 

CHARACTERIZATION OF THE PATHOGENESIS OF AMELANOSIS IN THE 
SMYTH LINE CHICKEN: A MODEL OF THE HUMAN AUTOIMMUNE DISEASE 

VITILIGO 



By 

EDMUND C. LEUNG 

May 1998 

Chairman: Wayne T. McCormack, Ph.D. 

Major Department: Immunology, Pathology and Laboratory Medicine 

Autoimmune diseases result when components of normally innocuous body 
tissues have unexpectedly undergone changes that make them appear foreign to the 
immune system. The immune system recognizes aberrantly expressed self proteins as 
nonself and recruits an immune attack on self. 

In the human autoimmune disease vitiligo, the pigment producing cells of the 
skin, the melanocytes, are destroyed and patients manifest irregular expanding 
depigmented patches of skin. An animal model for vitiligo, the Smyth line chicken, 
displays a spontaneous loss of feather and ocular melanocytes and the feathers 
progressively become whiter. Fifty percent of the birds also become blind. 

By adoptive transfer of splenocytes, I demonstrated that amelanosis can be 
transferred and mediated by lymphocytes. This is the first direct demonstration that 
lymphocytes mediate the disease. Prior research by bursectomy has shown antibody 
mediation. A role for y5 T cells was previously suggested by their expansion in number 



xn 



of peripheral blood lymphocytes with age in SL chickens. Sequence analysis of the T cell 
receptor y5 repertoire of the peripheral blood lymphocytes indicated a polyclonal 
expansion, rather than monoclonal or oligoclonal, a result that might be expected if they 
played a direct role in antigen-driven pathogenesis. The expansion of y8 T cells may be 
secondary to spillover from the site of inflammation at the regenerating feather pulp. A 
future experiment to examine the T cells found in the developing feather may 
demonstrate recurrence of an oligoclonal subset of T cells. This could lead to the 
development of therapy aimed at inhibiting clonal T cell activation. 

Southern blot analysis indicates that depigmentation does not appear to show an 
association with the presence of integrated endogenous viruses. The genetic heterogeneity 
present in SL chickens and revealed by the ev genotyping shows the feasibility of genetic 
linkage mapping to find vitiligo susceptibility loci. 

There are many diseases that have autoimmune responses to what are normally 
innocuous everyday proteins produced in the body. Studying autoimmune vitiligo adds 
one more piece to the autoimmune disease puzzle. One to two out of every 100 persons 
suffer from vitiligo. If a common factor can be found then preventive therapies would 
enhance the lives of many people. 



xin 



CHAPTER 1 
INTRODUCTION 

Autoimmunity 

The normal function of the adaptive immune response to a foreign antigen is the 
clearance of the foreign antigen (Ag) from the body. This is mainly achieved through the 
B lymphocyte compartment, which develops in the marrow in mammals or the bursa in 
avian species, and through the T lymphocyte or thymocyte compartment that matures in 
the thymus. The body has learned to distinguish between self-antigens and foreign 
antigens during T lymphocyte ontogeny. Essentially self-tolerance is established before 
mature T cells leave the thymus to enter the peripheral circulation for normal 
surveillance. Tolerance can be induced to some foreign antigens and self-antigens in the 
periphery by several means. Antigen presenting cells (APCs) which include B cells and 
various mononuclear cells (monocytes in blood, macrophages in tissues, Langerhans cells 
in the skin, Kupffer cells in liver, dendritic cells in lymph nodes) process and present 
antigen peptides in the groove of the major histocompatibility complex (MHC) 
molecules. 

The Ag-MHC complex engages with the T cell receptor heterodimer on the 
surface of a naive T cell migrating through the cortical regions in lymphoid tissue. Co- 
stimulation is provided by B7 on the APC engaging CD28 on the T cell. The activated T 
cell then remains in the lymphoid tissue, proliferates and differentiates into armed 

1 



effector T cells. T helper 1 (Thl) cells will instruct phagocytes to clear involved tissue 
cells containing intracellular parasites; Th2 cells will activate the corresponding B cell in 
the germinal centers to multiply, undergo somatic hypermutations (affinity maturation), 
differentiate to become plasma cells and secrete antibodies to complex the extracellular 
antigen or to opsonize foreign particles for recognition by phagocytes. T helper cells will 
also activate cytotoxic (CTL) T lymphocytes to destroy infected cells. The goal is 
complete and efficient clearance of the antigen from the body. Memory B and T cells are 
generated in preparation for a second exposure to the antigen with B cells undergoing 
somatic mutation adding diversity and better specificity. This is a general description of 
an ideally functioning immune system (reviewed in Janeway and Travers, 1997; Abbas et 
al, 1991). 

Inappropriate responses by T cells have been suggested to initiate autoimmunity 
as a result of a sustained immune response against self-antigen. Inappropriate T cell help 
can activate a harmful antibody response against self-antigens and activate 
polymorphonuclear cells (PMNs) to cause tissue damage. T helper cells will recruit 
cytotoxic T cells. Autoimmune antibodies can bind to the target surface antigen and 
cause complement-mediated cytolysis of the self tissues. Autoimmune antibodies can also 
initiate antibody dependent cell-mediated cytotoxicity (ADCC), recruiting natural killer 
cells to perform cytolytic killing of the autoantigen-expressing target cell. 

Autoimmune responses can be described as a loss of self-tolerance. If there is a 
sustained immune response that develops against the self-antigens, it becomes chronic if 
the initial immune effector mechanisms can not eliminate the antigen completely. The 
fact that the body is constantly producing and is therefore providing a constant source of 



the very antigen that the immune response is against makes it nearly impossible for the 
vicious cycle to end. The immune response intended to protect the body from foreign 
intruders has in the case of an autoimmune disease launched a chronic inflammatory 
injury against its own tissues and may in some cases prove to be lethal. 
Tolerance Mechanisms 

Autoimmunity occurs as a result of a breakdown of tolerance mechanisms. 
Tolerance is achieved when T cells recognize antigen in the absence of co-stimulation but 
remain inactivated having received only one of the two required activation signals. 
Tolerance is established in peripheral tissues and maintained mainly by developing 
central tolerance of the developing thymocytes to self-antigens prior to exposure to the 
outside environment, and then by peripheral tolerance mechanisms. Bone marrow- 
derived precursor T cells undergo the process of thymocyte education by first positive 
selection for cells that respond to self MHC expressed on cortical epithelial cells found in 
the thymus. Only those that bind the self MHC molecules with some affinity but not 
excessively continue to the thymic medulla for negative selection. Those that do not bind 
or bind to self MHC too well are deleted. In negative selection, the thymic medulla 
presents self-antigen on self MHC molecules as expressed on bone-marrow derived 
dendritic cells and macrophages. Only those thymocytes that bind with some affinity but 
not too excessively are allowed to leave the thymus as competent mature T cells; the rest 
are deleted by inducing apoptosis. So potentially self-reactive T cell clones are deleted 
before joining the peripheral T cell repertoire. This establishes central tolerance. This has 
been demonstrated in mice expressing the Mis" (minor lymphocyte stimulating) gene 



product, which can interact with a high proportion of T cells expressing particular Vp 
gene segments (Vp8.1 subfamily; Vp6). Those T cells that recognize both Ms* and the 
appropriate H-2 allele were eliminated during T cell development in the thymus. Ms is 
considered a superantigen (Kappler et al., 1988; MacDonald et al., 1988). Developing 
thymocytes that bind viral or bacterial superantigens integrated into human and mouse 
genomes of some strains have likewise experienced intrathymic deletion rather than 
anergy or peripheral suppression to provide tolerance. 

Central tolerance is incomplete because not all self-antigens will be expressed in 
the thymus during T cell development. Therefore peripheral self-tolerance must be 
established. In the periphery, the mature T cell is activated by recognizing the 
peptide:MHC complex on a professional antigen presenting cell (APC) that must also 
provide costimulation of the T cell's CD28 receptor by B7 molecules on the APC. In the 
absence of co-stimulation, specific antigen recognition leads to anergy or deletion of the 
mature T cell. Those antigens expressed uniquely by peripheral organs will not normally 
induce clonal deletion unless transported to the thymus in sufficient amounts or brought 
to lymphoid tissue. This is especially the case for organ-specific intracellular self- 
antigens located in sequestered sites (Barker and Billingham, 1972) or self-antigens 
expressed below a minimal concentration level (Schild et al, 1990; Ferber et al., 1994). 
These are immunologically ignored because of the absence of co-stimulator activity on 
tissue cells. The autoreactive T cells specific for these self-antigens are usually not 
eliminated nor anergized unless the self-antigens become presented by professional APCs 
in lymphoid tissue that would break the ignorance (Aichele et al., 1996). 



In the B lymphocyte repertoire, elimination of potentially self-reactive B cells 
occurs when immature B cells bind to multivalent membrane bound self-antigens during 
B cell ontogeny within the bone marrow. They are deleted by apoptosis by activation of 
the Fas receptor. This is unlike mature B cells, which become activated by multivalent 
polyclonal foreign antigens and CD4 T cell help. Self-reactive immature B cells may be 
rescued in the bone marrow by gene rearrangement events editing and deleting the 
sequence encoding for an autoreactive receptor to that of a different specificity that is 
tolerant (Cornall et al., 1995). B cells in the preimmune repertoire may be excluded in 
the competition for follicular niches in lymphoid organs if the self-reactive B cell binds 
soluble or low avidity autoantigens. They are not selected to proliferate and die out in the 
T cell zone. Such excluded B cells can be rescued if they enlist T cell help. 

There are mechanisms that help mature B cells become tolerant to self-antigens in 
the periphery. B cells that recognize a self-antigen as they would a foreign antigen would 
enter the T cell zone of lymphoid tissue. However, there would not be appropriate 
antigen-specific armed CD4 + T cells to activate the B cells. Such T cells would not have 
been presented the appropriate antigen from an APC and would be absent from lymphoid 
tissues; thus, they would not be available to provide secondary stimulation to the B cell. 
Those B cells with self-antigen would end up undergoing apoptosis, although death can 
be delayed by expression of bcl-2. A second mechanism is seen in naive B cells just 
entering the periphery. Chronic exposure to the specific soluble autoantigen, such as 
soluble lysozyme, will cause them to downregulate surface IgM expression and the 
signaling pathways of activation in order to survive. These become anergic because they 
fail to generate CD28-dependent T-cell help. Normally, T cells would induce Fas- 



6 

mediated apoptotic deletion of the anergized B cell when they present autoantigen. 
However, in mice in which the B cells carry the Fas mutation Ipr, the B cells are not 
eliminated, nor can T cells deficient in the Fas-ligand, gld, trigger the apoptosis of the B 
cells. These mice have autoimmune accumulations of lymphoid cells (Cornall et al., 
1995). 

B cell tolerance can be induced depending on antigen dose. High doses of antigen 
may overwhelm the surface immunoglobulins of B cells and induce specific 
unresponsiveness. This helps maintain tolerance to abundant self-proteins like plasma 
proteins. Very low doses of antigen in which the density of peptide:MHC complex on 
APCs may be too low to be recognized (that is, below the recognition threshold) by the T 
cells that do encounter them. Depending on the MHC genotype of an individual, some 
rare proteins contain peptides that may be presented at levels that are sufficient for T cell 
recognition but will not induce activation or tolerance. Such T cells are immunologically 
ignorant. They would then not be able to stimulate a B cell. T cell tolerance can be 
demonstrated in bone marrow chimeric animals during fetal development studies (Abbas 
et al., 1991). If allogeneic bone marrow is donated before the host achieves immune 
competence, then the developing T cell precursors would undergo central tolerance to 
antigens of both host and donor origin, thus tolerating self-peptides presented by both 
MHC genotypes. 

In summary, central tolerance is established by clonal selection, strength and 
quality of antigen receptor signaling of the B cells, avidity of immature T cell receptors 
for the MHC-peptide complex, and apoptosis of deleted cells. Peripheral tolerance 
depends upon the need for co-stimulation by appropriate APC. 



Loss of Immununological Tolerance 

When the immune system is unable to remain unresponsive to self-molecules 
tolerance is broken and autoimmunity may result. There are organ-specific diseases and 
systemic autoimmune diseases. Two hypotheses suggest that autoimmunity arises due to 
defects in the establishment of central or peripheral tolerance, or a conventional immune 
response occurs against self-antigens that, under normal circumstances, did not need to 
establish tolerance and the tolerance became broken. As suggested by Lehmann et al., 
(1993) a circumvention by the display of previously cryptic host determinants to which 
the host never had the need to develop tolerance causes autoimmune recognition. 
Mechanisms of Autoimmunity 

Autoimmunity may develop against self-antigen for a number of possible reasons. 
As mentioned above there may be incomplete deletions of self-reactive clones due to 
immunologic ignorance. This ignorance (clonal escape) may be due to the differences in 
genetic susceptibility based on the differences in ability of different alleles of MHC 
molecules to bind and present autoantigens to autoreactive T cells. Thus, in healthy 
individuals, there are probably autoantibodies that have been characterized as consisting 
of unmutated germline sequences with low avidity for autoantigens, and there are antiself 
T cells (Schwartz, 1993). 

The genetics of autoimmune diseases has been demonstrated in many ways. The 
HLA haplotype has often shown associations for susceptibility. For example in diabetes, 
people who express the MHC class II alleles HLA-DR3 or DR4, which are tightly linked 
to the HLA-DQ genes (the relevant disease susceptibility genes), have a noticeably higher 



8 

frequency of disease (Todd, 1995; Vyse and Todd, 1996). In HLA-DQpl, the normal 
Asp-57 is substituted by an uncharged amino acid residue destabilizing the DQ molecule. 
Other genetic factors influence susceptibility. Identical twins have a higher frequency of 
having the same autoimmune disease than MHC-identical fraternal twins. The hormonal 
status of an individual affects disease susceptibility. Many autoimmune diseases show a 
strong sex bias. Diabetes in the NOD mouse is more severe and occurs at a quicker onset 
in the female (Wicker et al., 1986). Peak incidence of autoimmune diseases that are 
more common in females occurs during the child-bearing period. 

If antigens are expressed selectively in a specific tissue rather than ubiquitously 
throughout the body, such antigens would be less likely to have induced clonal deletion 
of autoreactive T cells in the thymus during T cell ontogeny. Antigens of peripheral 
tissues especially sequestered behind anatomical barriers would not come in contact with 
the developing T cell repertoire. Tissue cells do not express co-stimulatory molecules. 
However, tissue damage may occur as a result of sustained direct attack of the cells 
expressing the self-antigen, from immune-complex formation, or from local 
inflammation. These antigens then become newly available as neoantigens in the 
periphery and subject to immune scrutiny by T cells that had escaped deletion. An 
example is seen in systemic lupus erythematosus (SLE). A broad range of autoantibodies 
is produced against intracellular nucleoprotein components: nucleosomes, DNA, histones, 
and ribosomes. Immune complexes continuously can deposit on the renal glumeruli, 
joints, and small arteries, and subsequently recruit macrophages to try to eliminate these 
immune complexes in a never ending battle (Kotzin, 1996; Schwartz, 1993). 



Autommunity may be the circumvention of self-tolerance by the induction of 
responses to cryptic determinants to which the host was never made tolerant (reviewed by 
Lehmann et al., 1993; Sercarz and Datta, 1994). The responses are by members of the 
self-reactive repertoire that had evaded negative selection. Changes occur causing 
determinant spreading and the availability of neoantigens to induce activation of 
autoreactive T cells out of naive T cells. Additional self-determinants previously hidden 
from recognition now prime other previously naive T cells with additional specificities. 
Intracellular as well as extracellular proteins can be presented on class II and class I MHC 
(Moreno et al., 1991; Nuchern et al., 1990), leading to a wide range of newly available 
self-antigens. Intermolecular spreading (antigenic spread from one determinant/epitope to 
many in the same protein) and intermolecular spreading (antigenic spread from one 
protein to another) recruits more self-antigens newly available for the immune system to 
respond to. Endogenously produced antigens have been presented by APCs as newly 
recognized autoantigens on activated thyroid epithelia, hepatocytes and pancreatic cells 
(Dayan et al., 1991; Barnaba et al., 1989; de Berardinis et al., 1988). 

In NOD mice, autoimmunity seems to start against glutamate decarboxylase 
(GAD) and then by determinant spreading more antigens, such as insulin, have become 
target antigens (Tisch and McDevitt, 1996). GAD is produced by the pancreatic islet p 
cells. Recent evidence is the finding of an 18 amino acid peptide showing a high 
sequence homology between human GAD and the Coxsackie virus P2-C protein 
(Kaufman et al., 1992). This is an example of molecular mimicry as a result of a 
misdirected immune attack. 



10 

In response to an environmental change, cytokines, such as TNF-a and IFN-y 
(Pestka and Langer, 1987), may cause shifts in the peptides synthesized, and oxygen 
radicals have induced the heat shock protein (HSP) response. HSP have facilitated 
peptide binding onto the MHC (De Nagel and Pierce, 1992), and caused differences in 
self-peptides produced during stress. In the EAE model of T cell-mediated 
autoimmunity, Lehmann and colleagues (1992), showed that a single determinant of 
myelin basic protein (MBP), the peptide Acl-1 1, was the immunodominant determinant 
in the primary response to MBP. Other determinants were cryptic, although available. 
Later in the chronically diseased mice the formerly cryptic host peptide determinants 
became the immunodominant primers of the second immunization. This has 
demonstrated diversification of the T cell repertoire due to determinant spreading. 

Prior infections causing tissue damage and the inability to clear immune 
complexes have been suggested in the induction of autoimmune disease. There are 
several mechanisms that have been postulated to explain how viral involvement leads to 
autoimmunity (Aichele et al., 1996; Nakagawa and Harrison, 1996; Barnaba, 1996). 
Viruses are involved in the generation of new epitopes (neoantigens) causing a loss of 
tolerance (breaking of immune ignorance). Goverman and associates (1993) developed a 
transgenic mouse to mimic the spontaneous induction and pathology of multiple 
sclerosis, which expressed a TCR specific for myelin basic protein. Spontaneous EAE 
could not develop in a sterile environment, but it could develop easily if the mice were 
given pertussis virus alone or even simply housed in a nonsterile facility (Goverman et 
al., 1993). Anti-viral immune responses may shift and recognize shared molecular 



11 

components in self-antigens which may have altered expression in infected tissue. Thus 
antibodies trigger cross-reactive autoimmune reactions to shared determinants of the 
self-antigens (molecular mimicry) (Douvas and Sobelman, 1991). In rheumatoid arthritis, 
the HLA-DR pi alleles, which contain the QKRAA amino acid sequence in the CDR3 
region, have been associated with the autoimmune condition. QKRAA sequences as 
expressed by Epstein-Barr virus have been found in RA patients with enhanced humoral 
and cellular responses (La-Cava et al., 1997). 

Viruses have developed means to circumvent the host. As mentioned before, 
viruses may act as superantigens such as the Mis locus for T cells with certain Vp genes, 
or they may provide generalized immunosuppression, such as during HIV infection. The 
respiratory syncytial virus induces interferon to inhibit a proliferative response by human 
PBMCs (Preston et al., 1995). A T cell polyclonal activation by a bacterial superantigen 
could likewise overcome tolerance, as in rheumatoid arthritis or in EAE in which T cell 
clones expressing certain Vp genes all become activated. The bacterial superantigen 
staphylococcal enterotoxin B (SEB) activates Vp8 + T cells that engage the amino-terminal 
epitope of myelin basic protein. SEB induces relapse of the paralysis in mice that are in 
clinical remission and triggers paralysis in mice with subclinical disease after initial 
immunization with the Acl-11 epitope or after transfer of encephalitogenic T cell lines 
(Brocke et al., 1993). Thus incomplete deletions of self-reactive clones or aberrant 
stimulation or regulation of normally anergic clones later become newly elicited self- 
reactive clones. 



12 

Viruses can produce proteins that can regulate or counteract the antiviral 
responses of the host (Gooding, 1992; Marrack and Kappler, 1994). Epstein-Barr virus 
stimulates the conversion of uncommitted T helper cells into Th2 helper cells by the 
product of the BCRF1 gene which has structural and functional homology to IL-10. This 
allows EBV to prevent the induction of Thl -activated inflammatory responses initiated 
by such cytokines as IL-1, tumor necrosis factor, and interferon-y. The herpes simplex 
virus induces the infected cell to express HSV-Fc receptor, a heterodimer of 
glycoproteins E and I, which binds to the Fc region of the host's nonimmune IgG. This 
binding prevents complement-mediated lysis of infected cells by blocking access to the 
cell surface of antiviral antibody or effector cells (Bell et al., 1990). Cowpox virus codes 
for a soluble glycoprotein that has amino acid homology to that of the IL-1 receptor. 
This product probably competes with cell-bound IL-1 receptors for secreted IL-1, 
interfering with the activation of IL-1 cytokine-mediated inflammatory responses. 

In persistent infections, such as in autoimmune hepatitis, the infected tissue is 
destroyed during long-term chronic inflammatory responses to the replicating virus itself, 
or is destroyed by the cytotoxic T cell response to the viral antigens presented on the 
target tissue. This destruction inadvertently and continuously releases large quantities of 
the organ's self-antigens (especially those never exposed extracellularly) which become 
presented by professional APCs in lymphoid tissue (Koziel et al., 1992; Cerny et al., 
1994). Wounds may also disrupt tolerance by causing the release of self-antigens 
normally protected. 



13 

Endogenous viruses can also deregulate the expression of normal gene products. 
By their integration into the chromosome of the host, they cause interruptions in the 
normal functions of the genes and their protein products. Endogenous viruses can 
inactivate genes by premature termination of protein synthesis due to the addition 
nucleotides encoding a stop codon. Integration of endogenous viruses can create 
mutations that enhance transcription, and may therefore cause chronic protein expression. 
More about endogenous viruses will be discussed in Chapter 4. 
Regulation of Autoimmune Responses 

Immune regulation can either encourage the initiation of autoimmunity or act to 
maintain the tolerance. Cyclosporin is a potent suppressor of graft versus host disease 
(GVHD) and autoimmune diseases, including the suppression of amelanosis in the Smyth 
line chicken (Pardue et al., 1987). However, cyclosporin has also been shown to induce 
autoimmunity (Sorokin et al., 1986). 

Adhesion molecules and cytokines can affect autoimmune processes. In the EAE 
model for multiple sclerosis, transforming growth factor (TGF)-(i provided protection 
when the injection occurred for the period of 5-9 days after immunization with MBP; 
there was no protection if TGF-0 was administered before (days 1-5) or after (days 9- 
1 1). TGF-P is immunosuppressive to the Thl -produced interferon (IFN)-y in response to 
the presence of MBP (Santanbrogio et al., 1993). This Thl -mediated autoimmune 
disease was examined by Racke and colleagues (1995) for the role of co-stimulatory 
molecules. They demonstrated that in vitro activation of MBP-specific lymph node cells 
was inhibited by the combination of B7-1 and B7-2 activation. However in actively 



14 

induced disease, administration of anti-B7-l reduced disease; anti-B7-2 exacerbated 
disease. In murine diabetes, intercellular adhesion molecule 1 (ICAM-1) is involved in 
recruiting lymphocytes to the pancreatic islet cells. Cytokines interferon-y and tumor 
necrosis factor-a secreted by the islet cells could induce the ICAM-1 expression on 
pancreatic (5 cells, and immunointervention by anti-ICAM-1 and anti-LFA-1 mAbs would 
significantly prevent the development of diabetes (Yagi et al., 1995). Whereas the 
administration of cytokines promotes IDDM, the administration of mAbs against Thl- 
produced cytokines blocks the development of the disease (Song et al., 1996; Mossman 
and Coffman, 1989; Maclaren and Atkinson, 1997). 

Oral tolerance has been a method of antigen-specific immunotherapy for 
autoimmune disease (reviewed by Hafler and Weiner, 1995; Muir et al., 1993). The use 
of low doses of orally administered autoantigens is suggested to utilize the secretion of 
downregulatory cytokines such as TGF-P and the Th2 responses of IL-4 and IL-10 to 
cause active suppression. High dose therapy induces anergy, the unresponsiveness of 
Thl function in a systemic presentation of autoantigen. Intermittent injections of the 
autoantigen insulin, or the B chain of insulin in incomplete Freund's adjuvant, induces an 
active suppressive response that induces a protective insulitis in the NOD mouse model 
of diabetes (Muir et al., 1995). 
Autoimmune Diseases Cause by Antibodies 

Autoantibodies may bind to autoantigens on the cell surfaces or extracellular 
matrix and initiate tissue damage similar to type II hypersensitivity. By interaction of 
the bound antibody with Fc receptor-bearing macrophages, there is increased clearance of 



15 

red blood cells in autoimmune hemolytic anemia. These IgG- or IgA-coated cells may 
fix complement to lyse these RBCs. The binding of autoantibodies to cells in tissues 
allows for the fixation of sub-lytic doses of the membrane attack complex of complement 
proteins to stimulate an inflammatory response recruiting inflammatory 
polymorphonuclear cells and natural killer mediated antibody-dependent cell cytotoxicity 
to cause tissue damage. An example of this is seen in Hashimoto's thyroiditis. 
Autoantibodies binding to a cell surface receptor can cause excessive activity by the 
receptor or inhibit its stimulation by its natural ligand. Patients become hyperthyroid in 
Grave's disease because antibodies to thyroid stimulating hormone prevent normal 
feedback to the production of thyroid hormone. Antibody response to soluble antigens 
produces immune complexes that are normally cleared by red blood cells, which have 
complement receptors, and phagocytes, which have complement and Fc receptors. 
Failure to clear immune complexes leads to persistent presence and deposition, especially 
after tissue injury continues to generate more of the antigen as in serum sickness, and 
chronic infections such as bacterial endocarditis, and systemic lupus erythematosus. In 
SLE, antibodies are formed against ubiquitously found intracellular nucleoproteins of all 
nucleated cells, such as DNA, RNA, and histones. Immune complexes are formed that 
deposit on the walls of small to medium blood vessels, especially in the renal glomeruli. 
These complexes attract complement and PMNs, causing more tissue damage and starting 
the cycle again. SLE is considered therefore a systemic rather than an organ-specific 
autoimmune disease. 

Mothers pass on their IgG antibodies to the fetus through the placenta. Babies 
born to mothers with IgG-mediated autoimmune diseases may often show the symptoms 



16 

similar to that of the mothers temporarily, until the baby starts manufacturing its own 
antibodies. This describes one form of passive adoptive transfer of autoimmunity. 
Autoimmune Diseases Caused by T Cells 

Autoimmune T cells may also be directly involved in tissue destruction or in 
causing inflammation by activating macrophages, as well as being necessary to maintain 
autoantibody responses. They require the autoantigen presented on MHC with co- 
stimulatory ability from a professional APC and at sufficient quantities to interact in 
lymphoid tissue in order to initiate an autoimmune response. In insulin-dependent 
diabetes mellitus, the insulin-producing (3 cells of the pancreatic islets are selectively 
destroyed by CD8 + T cells, which have received inappropriate activation from CD4 + T 
cells that were activated by APCs. The specificity of the autoantigen as the target of 
destruction can be seen in pancreas transplants when, even though the graft is from an 
identical twin donor, the recipient's T cells destroy the graft. 

Vitiligo in Humans 

Vitiligo is an acquired melanin pigmentary disorder of the epidermis and hair 
follicles, manifested by expanding, irregular, depigmented lesions of the skin. Vitiligo 
can appear at any stage of life (LePoole et al., 1993) but half of those affected develop 
vitiligo before the age of 20. Vitiligo is a common disease, affecting 1-2% of the 
population in all racial groups worldwide. It is otherwise asymptomatic and most 
patients remain physically in good health. However it does predispose affected persons 
to sunburn skin damage and an 180-fold increased risk of melanoma (Dunston and 
Haider, 1990). The often severe cosmetic disfigurement has psychological effects and 



17 

current treatment modalities for vitiligo, such as phototherapy with psoralens and high 
intensity UV-A irradiation (PUVA), are difficult, expensive, and usually disappointing 
(Grimes, 1993). 
Melanocyte Biology 

Melanocytes, which are located in the epidermis of the skin, produce the pigment 
melanin, and release the melanin to keratinocytes. The biosynthesis of melanins occurs in 
melanocytes, and the enzyme tyrosinase catalyzes several of the initial steps of 
melanogenesis, which occurs within the melanosome organelle of the melanocyte (Orlow 
et al., 1993; Prota, 1988; Bennett, 1993). This includes the hydroxylation of tyrosine to 
dopa; the oxidation of dopa to dopaquinone and intermolecular circularization and 
oxidation of dopaquinone to dopachrome. Divergent paths then take place. In the 
presence of metals, dopachrome eventually becomes 5,6 dihydroxyindole, which 
ultimately undergoes oxidative polymerization to create eumelanin. In the presence of 
cysteine, the sulfur-containing phaeomelanins and trichochromes are polymerized. The 
black eumelanins are insoluble in all solvents and the phaeomelanins, the browns and 
reds, are alkali-soluble. 

Two of the enzymes involved in melanin biosynthesis are characterized in studies 
using mutations in the mouse at the albino locus, which encodes tyrosinase, and the 
brown locus, which encodes tyrosinase related protein- 1 (TRP-1). Trp-1 has a 43% 
identity to tyrosinase at the protein level. Due to two amino acid substitutions, the 
homozygous b/b mouse produces brown melanin which at physiologic pH, is soluble 
instead of the black melanin produced in the wild type which is insoluble. During 



18 

melanin synthesis, TRP-1 appears present only in unmelanized stage I and stage II 
melanosomes, and tyrosinase is primarily found in late stage III and IV melanosomes. 
Both are found in the Golgi and trans-Golgi and then enter a LAMP- 1 -positive (a marker 
for organelles of the endosomal-lysosomal lineage) organelle that is consistent with a late 
endosome (Orlow et al., 1993). The mature melanosomes travel along the melanocyte 
dendritic processes from which they are transferred to the keratinocyte for depositing. 
Vitiligo Pathology 

There are several theories to explain the etiology of vitiligo, including self 
destruction of the melanocyte, the neurogenic, the immune, and the genetic (Ortonne and 
Bose, 1993; Ortonne et al., 1983). Vitiligo is characterized by inherent melanocyte 
defects, loss of melanocytes accompanied by T cell infiltration in the affected tissue (Le 
Poole et al., 1993b; Hann et al., 1992; Badri et al., 1993; Erf et al., 1995b), disturbances 
in peripheral blood lymphocyte subpopulations (Mozzanica et al., 1990; Abdel-Nasser et 
al., 1992; Erf et al., 1995a), and the presence of serum autoantibodies directed against 
melanocyte antigens (Harning et al., 1991; Austin et al., 1992; Searle et al., 1993). Park 
et al. (1996) suggest that the antibodies are directed primarily against a 65 kDa antigen. 
Both antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated 
damage have been induced to cultured human melanocytes by anti-melanocyte antibodies 
from the sera of vitiligo patients (Norris et al., 1988). This suggests a possible role for 
autoantibodies in vitiligo. Two groups have demonstrated that the antibodies in the serum 
of vitiligo patients is against tyrosinase (MW of 70 kDa) an enzyme involved in the 
biosynthesis of melanin by the melanocytes (Fishman et al., 1997; Song et al., 1994). 



19 

Enzymes have been known to be autoantigens in various autoimmune diseases; GAD is a 
major autoantigen in the NOD mouse model of diabetes (Maclaren and Atkinson, 1997). 

The melanocytes have been shown in vivo and in vitro to have intrinsic aberrant 
morphology, increased tyrosinase activity and increased acid phosphatase activity (Boissy 
et al., 1983, 1986), suggesting that an underlying melanocyte defect may predispose these 
cells to abnormal antigen presentation, which may be important for pathogenesis (Boissy 
et al., 1991). Abnormal presentation may be possible, considering the abnormal 
expression of class II HLA molecules by perilesional melanocytes in about 2/3 of the 
patients studied, as well as a six-fold increase in expression of ICAM-1 (Al-Badri et al., 
1993; Ann et al., 1994). The endoplasmic reticulum found in the melanocytes is 
irregularly dilated and circular rather than narrow and elongated, floccular material can be 
found within the cisterna (Boissy, 1991; Hafler, 1995; Im et al., 1994), and membrane 
bound compartments of melanosomes that contain autophagocytic activity possibly 
bound for lysosomal destruction have also been observed (Im et al., 1994). Yet it has not 
been proven that these melanocyte defects actually are toxic. 

The presence of an inflammatory rim of cellular infiltrates detected in 
inflammatory vitiligo skin coincided with the loss of melanocytes, and infiltrating T cells 
in the epidermis were frequently juxtaposed to the remaining melanocytes. This rim of 
cellular infiltrates was in the perilesional skin in the basal layer of the epidermis and with 
the destruction mainly by CD8 + T cells. These melanocyte abnormalities are present prior 
to the presence of mononuclear infiltration (Boissy et al., 1983, 1986). Keratinocytes 
may contribute to the HLA-DR class II presentation of melanocyte antigens following 
phagocytosis of melanosomes within the destroyed melanocytes (Le Poole et al., 1996). 



20 

Immunohistochemical studies have shown the actual loss of melanocytes 

(LePoole et al., 1993). Melanocyte loss is accompanied by epidermal and dermal 

lymphocyte infiltrations in the active lesions (Harm et al., 1993; Badri et al., 1993) with 

increases in both CD4 + and CD8 + T cells (Hann et al., 1993). Cellular infiltrates have IL2 

and IFN-y expressed, indicating a possible Thl recruitment (Abdel-Nasar et al., 1994). 

The Association of Vitiligo with Other Autoimmune Diseases and the Genetics of 
Vitiligo Susceptibility in Humans 

Vitiligo is inherited as a polygenic trait and probably involves mutations in at 
least 3 or 4 autosomal recessive genes (Lacour and Ortonne, 1995; Bhatia et al., 1992). 
The risk of developing vitiligo appears to be strongly dependent on one's kinship to the 
proband and not dependent on gender. The relative risks, whether parent, sibling or 
offspring of probands, show considerable variation, pointing to a lack of involvement of 
a single gene with complete penetrance (Majumder et al., 1993). However, the high 
frequency of familial aggregation of the disease in association with other 
autoimmune/endocrine diseases, and the presence of organ-specific autoantibodies in the 
first and second degree relatives of the patients gives support to a genetic predisposition 
in vitiligo (Mandry et al., 1996). 

Recent data suggest that human endogenous viruses may be involved in the 
pathogenesis of a variety of human autoimmune diseases, such as diabetes, systemic 
lupus erythematosus, rheumatoid arthritis, psoriasis, and inflammatory neurologic 
diseases (Yoon, 1990; Urnovitz and Murphy, 1996). Vitiligo may indeed be triggered by 
a viral infection in select patients (Grimes et al., 1996). Affected vitiligo patients can 
also express the hypothyroidism found in Hashimoto's thyroiditis, Grave's disease, and 



21 

alopecia universalis (loss of hair). Thus vitiligo's association with other autoimmune 
diseases (Elder et al., 1981; Shong and Kim, 1991; Schallreuter et al., 1994a; Nath, 
1994) has categorized it within the Autoimmune Polyglandular Syndrome I diseases. 

There is evidence suggesting a neuronal involvement in the disease in order to 
explain the segmental and symmetric distribution of depigmentation found in some 
patients or why there is a lack of depigmentation below the level of spinal cord injury in a 
patient with transverse myelitis and vitiligo. In a study of dermal nerves in vitiligo 
patients, Al'Abadie and colleagues (1995) concluded that cycles of initial events of 
vitiligo may cause axonal damage with later nerve regeneration. They suggested that the 
destructive mechanism of melanocytes may be triggered by the neurotransmitters released 
by nerve endings which are of close proximity to the melanocytes. Studies of 
neuropeptide and neuronal marker immunoreactivity in skin biopsies such as 
neuropeptide Y (NPY), support the theory that there is neuronal involvement in vitiligo 
and that NPY may have a role in the pathogenesis of vitiligo (Al'Abadie et al., 1994). 

The C57BL/6J-vit/vit Mouse Model for Vitiligo 

Researchers normally gravitate to the mouse in order to identify an animal model 
that depicts the disease condition in the human. Such exists in the C57BL/6J-vit/vit 
mouse model for vitiligo, which progressively loses much of its epidermal and follicular 
pigment cells during successive shedding of fur. Eyes are also affected (Lerner 1986; 
Lamoreux et al., 1992). This has been mapped to a recessive allele of the microphthalmia 
gene locus (/w' v ") (Halaban et al., 1993; Lamoroux et al., 1993). The mi gene has been 
identified as a member of a basic-helix-loop-helix zipper transcription factor family 



22 

(Hodgkinson et al., 1993) able to bind transcriptional control elements in melanocyte- 
specific genes. This mf* allele has a single G-to-A transition, causing an Asp222Asn 
substitution in the first helix domain (Steingrimsson et al., 1994). The mf" gene product 
of the mouse and the Mitf equivalent in the human regulate the expression of melanocyte- 
specific genes including TRP-1 and TRP-2 (Bertolotto et al., 1996; Yasumoto et al., 
1997). However, this strain of mice does not exhibit an autoimmune component 
comparable to what is seen in the human, and the affected tissues fail to show a 
lymphocyte infiltration (Lerner et al., 1986; Boissy et al., 1987). The premature death and 
cytological aberrations found in this strain is considered to be the consequence of an 
innate cellular defect; it has been concluded that the depigmentation is the result of a 
genetic defect that is not initiated by a systemic or local condition (Im et al., 1994) and so 
it is not a suitable model for human vitiligo studies. 

The Smyth Line (SL) Chicken Animal Model for Vitiligo 

The Smyth line chicken represents a good animal model for the study of human 
vitiligo (reviewed by Smyth et al., 1981; Smyth, 1989). SL chickens are characterized by 
a spontaneous loss of feather and ocular melanocytes beginning around 6-8 weeks 
posthatch (Figure 1-1 and 1-2); thus, feathers progressively become whiter rather than 
maintain the original brown feather color of the parental Brown line strain (Figure 1-3). 

The progenitor of the Smyth line was a spontaneous amelanotic female hatched in 
1971 from the Massachusetts Brown line, and since then, a current frequency of 
approximately 1-2% of the Brown line spontaneously becomes amelanotic. From 



23 




Figure 1-1. A female Smyth line chicken displaying amelanosis of stage 4 




Figure 1-2. A group of Smyth line chickens at various stages of amelanosis. 



24 




Figure 1-3. A typical pair of parental Brown line chickens. 



25 



outcrosses to various other chicken lines and backcrosses of the original mutant back to 
the Brown line, the delayed amelanosis (DAM) line, later renamed the Smyth line, was 
developed by selection for onset of amelanosis and severity (Smyth et al., 1981). Since 
the fifth generation, the Smyth line has closely resembled the parental line and three 
novel MHC B alleles are segregating in both lines (Erf et al., 1995a). 

Pigment and melanocyte loss in SL chickens can range from partial to complete 
amelanosis, and about 50% of depigmented birds are blind due to melanocyte destruction 
in the choroid and retinal pigmented epithelium (Smyth, 1989). The magnitude of 
amelanosis in any Smyth line bird depends on the time frame of each feather's 
development when melanin synthesis and pigment deposition are destroyed. Up to 90% 
of a hatch will exhibit the amelanotic phenotype as reported by Smyth (Smyth, 1989) 
although as described in chapter 2, only about 60% are amelanotic in the University of 
Florida colony. 

SL chickens also have an increased incidence of thyroiditis/hypothyroidism 
resembling human Hashimoto's thyroiditis, and a defeathering defect analogous to human 
alopecia (Smyth, 1989). Thus it is characterized by the same features as human vitiligo, 
including an association with other autoimmune diseases (Elder et al., 1981; Schallreuter 
et al., 1994; Shong et al., 1991). SL melanocytes have been shown in vivo and in vitro to 
have intrinsic aberrant morphology, increased tyrosinase activity, and increased acid 
phosphatase activity (Boissy et al., 1983, 1986), suggesting that, as in human vitiligo 
(Boissy et al., 1991), an inherent melanocyte defect may be important for pathogenesis. 



26 



Melanocyte Biology 

As in the human, the melanin pigment is a product of melanosomes, the 
melanocyte cytoplasmic organelles that produce the pigment granules. The ocular 
melanocytes, as well as the choroid and anterior surface of the iris of the eye, originate 
from pleuripotent cells in the embryonic neural crest (Smyth et al., 1981; Smyth, 1989). 
The retinal pigmented epithelium and the iris are derived from the optic cup. The 
undifferentiated melanoblasts congregate as dermal reservoirs initially populating outside 
near the base of the growing feather pulp follicle (Figure 1 -4). Melanoblasts migrate 
through the feather pulp toward the periphery of the pulp and align near the basement 
membrane interface of the barbed ridges and the pulp (Figure 1-5). Dendritic extensions 
extend from the melanocytes to the barbule cells, where melanin granules are deposited in 
a situation similar to the keratinocyte in the human skin. After the barbule cells receive 
pigment, the melanocytes retract and degenerate (Smyth, 1989; Figure 1-6). 

In chickens, tyrosinase was the only known catalyst of melanogenesis (Smyth, 
1989). However, more recently, tyrosinase related protein, TRP-1, but not tyrosinase, has 
been detected by serum autoantibodies of Smyth line chickens (Austin et al., 1995). 
There are five tyrosinase isozymes each with a molecular weight of approximately 66 
kDa, and an additional nine other proteins that have been isolated from cultured chicken 
melanocytes and are assumed to be involved in melanogenesis. Dopa 
(dihydroxyphenylalanine) is one of the intermediate products produced by tyrosinase and 
can be used in tyrosinase detection (White, 1983). 



27 




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Amelanosis Pathology in the Smyth Chicken 

As in human vitiligo patients, the Smyth line chicken melanocyte is the target of 
autoimmune destruction, but instead of the skin and hair follicles, the feathers, the 
choroid, and retinal pigmented epithelium are affected. Both autoantibodies and cell- 
mediated immunity may be involved in the pathogenesis of vitiligo in the Smyth line 
animal model. Melanocyte-specific autoAb have been detected 1-4 weeks prior to 
depigmentation in SL chickens, as observed in humans, and the autoAb have been shown 
to recognize at least three melanocyte proteins between 65 and 80 KDa, which are 
localized to the melanocyte cytoplasm and plasma membrane (Austin et al., 1992). 
Because the enzyme tyrosinase is involved in the biosynthesis of melanin, and because 
enzymes are known to be autoantigens in other autoimmune conditions, tyrosinase has 
been suggested as a possible autoantigen for vitiligo. It was recently shown that Trp-1, 
the most immunogenic of the tyrosinase-related proteins, is a major autoantigen 
recognized by serum autoAb in SL chickens (Austin et al., 1995). 

The Smyth Line chicken demonstrates a functional immune system capable of 
providing an autoimmune response in the initiation and progression of melanocyte 
destruction. If the immune system is experimentally voided or suppressed then it might 
be expected that the amelanotic condition may be eliminated or reduced. When the B 
lymphocytes are essentially eliminated by neonatal bursectomy the effect is a decrease in 
the incidence and severity of amelanosis (Lamont and Smyth, 1981). Cyclosporin A 
treatment and corticosteroid-induced immunosuppression result in a decreased incidence 



31 

and severity of amelanosis in the Smyth Line chicken as long as the treatment continues. 
However, when the therapy is stopped the amelanosis will be as severe as littermate non- 
treatment controls (Boyle et al., 1987; Pardue et al., 1987). 

It appears that the MHC locus influences the disease progression. Three MHC 
alleles have been found segregating within Smyth line chickens and sublines have been 
established {B 101 , B 102 and B 103 ). Of the three, B 101 exhibits the earliest age of onset 
and the most severe phenotypes (Erf et al., 1995a). 

Evidence for T cell involvement in amelanosis in the Smyth chicken is less 
extensive. Studies have demonstrated that cyclosporin A, a potent inhibitor of IL-1 and 
IL-2 release that normally stimulate PBMLs and NK cells, can measurably reduce the 
incidence and severity of amelanosis in SL chickens (Pardue, 1987). There is also 
histological evidence of an intense T cell involvement in amelanosis of the SL chicken 
(Erf et al., 1995b). Lymphocytic infiltration is consistently seen associated at sites of 
melanocyte destruction (Smyth, 1 989) which have recently been shown to be primarily T 
cells (Erf et al., 1995b) as detected by polyclonal and monoclonal antibodies against 
functionally important surface T cell molecules (Cooper et al., 1991; Chen et al., 1988; 
Chen et al., 1989; Char et al., 1990; Lahti et al., 1988). 

T cells infiltrate growing feather pulp as much as 6 weeks prior to visible signs of 
vitiligo. Significantly greater numbers (9-14 fold) of T cells of all three subpopulations, y 
5 (detected by TCR1), Vpl + api (detected by TCR2), and Vp2 + ocp2 (detected by TCR3) 
are found present in cross sections of the feather pulp in SL as compared to BL prior to 
and throughout amelanosis. Of note are the proportions of TCR2 + cells being 



32 

significantly higher, and of TCR1 + cells being significantly lower, as compared to Light 
Brown Leghorn control birds (LBL, a related line with similar plumage but no incidence 
of vitiligo) (Erf et al., 1995b). 

Initially, both the SL and control BL have a CD47CD8 4 ratio close to 1 . Prior to 
and early in the amelanosis, CD4 + T cells are found histologically in a central, 
perivascular region within the confines of the feather pulp. As the disease progresses, the 
ratio decreases to below 0.4 (indicating mainly CD8 + cytolytic cells) but then rebounds 
to about 0.8 late in the disease (indicating an increase in CD4 + cells, probably to recruit B 
cells). Mainly CD8 + cells remain after the melanocytes have been destroyed (CD47CD8 + 
ratio of 0.3). The shift in the CD4/CD8 ratio from below 0.4 back to 0.8 in late disease 
suggests the activation of T helper 2 (Th2) cells involved in a humoral response to 
melanocyte autoantigens released during their destruction. In the later stages of 
amelanosis CD4 + cells become scattered throughout the pulp and surrounded 
melanocytes. CD8 + T cells are observed throughout the pulp and are most abundant near 
the epithelial barb ridges and associated with melanocytes (Erf et al., 1995b). This 
indicates that the CD8 + T cells have penetrated beyond the pulp to get to the melanin- 
containing barb ridges. 

A working hypothesis for the pathogenesis of amelanosis found in the Smyth line 
chicken is that there are inherent defects in the Smyth line melanocyte that cause the cells 
to self-destruct. These cells may self destruct or possess a quality that predisposes them 
to abnormal antigen expression. Abnormal presentation by the melanocytes themselves 
may target them for CTL-mediated destruction. This releases the internal components of 



33 

the cell as neo antigens that the immune system responds to by the production of 

autoantibodies. 

Genetics of vitiligo susceptibility in SL chickens 

Three SL sublines have been described, and each one is homozygous for a 
different MHC haplotype [B*™, B^2 and B^^ based on serological typing (Erf et al., 
1995a). While all three sublines are similar in incidence of vitiligo, the B*"* SL subline 
has the earliest age of onset, with more severe expression of vitiligo, and a greater 
incidence of blindness due to retinal dystrophy as compared to the B^2 and B*™ 
sublines. Interestingly, the three SL sublines also exhibit differences in the distribution of 
T cell subpopulations in peripheral blood as compared to the controls, LBL and Brown 
line (BL, the parental line from which SL was derived, with a 2% incidence of vitiligo). 
B^Ol SL chickens at 40 weeks of age contained significantly fewer CD4 + and TCR2 + ap 
T cells and significantly more TCRL y5 T cells in peripheral blood lymphocytes (PBL) 
of 40 week-old SL chickens (Erf et al., 1995a). This increase in PBL y8 T cells is 
detectable as early as 13-18 weeks of age (Erf and Smyth, 1996). Similar differences 
were found in the B^2 subline, but not in B^3 subline. 

Chicken Immunology 

Chicken Immunoglobulin genes and B cell development 

Antibodies or immunoglobulins (Igs) are the antigen specific receptors produced 
exclusively by the B lymphocytes. They bind soluble antigens (proteins, nucleic acids, 
polysaccharides, lipids, and small chemicals) by recognizing conformational determinants 
of the antigens in their native three-dimensional form as well as determinants unmasked 



34 * 

by denaturation or proteolysis. During the different phases of their maturation as an 
adult cell, the B cell provides both cognitive and effector functions for the humoral 
immune response. With their membrane Ig receptors, B cells recognize and respond to 
specific antigens. Through their MHC class II they present processed Ag to T cells. 
Following antigenic stimulation they become effector cells by releasing serum Ig as 
plasma cells. 

All progenitor B cells develop from pleuripotent stem cells that migrate from the 
embryonic thoracic aorta to the yolk sac, where the Ig heavy chain undergoes D-J 
rearrangement, and then colonize the spleen, yolk sac, and bone marrow. In these organs 
the cells rearrange the V H and then the V L genes, resulting in surface IgM expression. 
Between embryonic days 8 and 14 about 20,000 to 30,000 of these B cells start to 
accumulate in the bursa (Reynaud et al., 1987). 

Mammalian B cells rely on large numbers of germline Ig gene segments and the 
combinatorial diversity of Ig gene rearrangement to generate a diverse Ab repertoire, 
which occurs continuously throughout life, in the bone marrow. In contrast, chicken Ig 
genes undergo rearrangement of single functional V H and V L gene segments within a short 
time period during embryogenesis. Then diversity is generated in the rearranged variable 
regions by somatic gene conversion using a pool of pseudogenes as sequence donors 
(Reynaud et al., 1987; McCormack et al., 1993). 

Gene conversion provides a progressive substitution of the sequence within the 
functional V L or V H gene with sequence blocks donated or copied from the nonfunctional 
pseudogenes. Progressive overlapping replacement events efficiently corrects out of 
frame joints and expands the diversity (McCormack et al., 1993). After 6 months the 



35 

bursa involutes, and no new B cell development occurs. The B cell population is 
maintained by the proliferation of a post bursal population. 
Chicken T cell receptor genes and T cell development 

T cells recognize antigens that are linear processed fragments of foreign proteins, 
but only when presented to the T cell receptors (TCR) in physical association with a self 
MHC molecule expressed on the surfaces of syngeneic antigen presenting cells or on 
target cells. TCR are heterodimer plasma membrane proteins and the surfaces that bind 
the peptide-MHC complex are expressed as unique determinants, which differ in one 
clone from another, providing different antigen-MHC specificities. The particular TCR 
will recognize peptides associated with either class I MHC or class II molecules, which 
are also recognized by the CD8 or CD4 coreceptor molecules, respectively. 

Both TCR aP (50kDa) and yo (40kDa) receptor molecules are disulfide-linked 
heterodimer glycoproteins noncovalently associated with a CD3 complex as in mammals 
(Sowder et al., 1998; Chen et al., 1989; Char et al., 1990). They are identified by anti- 
TCR antibodies: y5 (TCR1) (Sowder et al., 1988), apl (TCR2) (Cihak et al., 1988; Chen 
et al., 1988), and <xp2 (TCR3) (Chen et al., 1989; Char et al., 1990). 

Chicken precursor T cells originate from pleuripotent stem cells in the embryonic 
thoracic aorta that then colonize the spleen, yolk sac, and bone marrow. Thymocyte 
progenitors enter the thymus in three waves into the thymic epithelium, which produces 
P2 microglobulin as a chemoattractant (Dunon et al., 1990). Each wave of progenitors 
will give rise to all three different forms of T cells, always in the order of y8, VpTaP, 
and Vp2 + ap (Figure 1-7). 



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37 



Chicken y5 T cells (TCR1) appear first in thymocyte development, as TCR y8 
/CD3 cells. They enter the thymus at embryonic day 6 (E6), avoid thymic education and 
selection, migrate quickly through the thymus without clonal expansion, reach peak 
levels by E15, and exit after day E15 (Cooper et al., 1991; Dunon and Imhof, 1996). y5 T 
cells do not express CD4 or CD8 and are not self MHC-restricted, as they do not undergo 
intrathymic selection (Raulet, 1989; Chen et al., 1996). In the periphery, the yd T cells 
reach 20-50% in the adult with a very high predominance in the intestinal epithelium and 
either absence or minor presence in chicken Peyer's patches and cecal tonsils (Bucy et al., 
1988). They are resistant to death by receptor cross linking and apoptosis (George and 
Cooper, 1990) and are not subject to arrest by cyclosporin A (Bucy et al., 1990). VpTocP 
cells, in contrast, are susceptible to receptor modulation and apoptotic death (Smith et al., 
1989). TCR1 cells are relatively dispersed and rarely form lymphoid nodules or 
aggregates even in the spleen or intestine. About two-thirds will express CD8 in the 
spleen and intestine (Chen et al., 1988; Bucy et al., 1988) but rarely in the circulation. y5 
T cells lack GVH potential and their proliferative response is relatively low (Sowder et 
al., 1988). 

The second wave of chicken T cells enters the thymus by El 2- 13 and this wave 
contributes predominantly to the Vpi + aP T cell subset. These do express initially CD4 
and CD8, undergo thymic maturation and clonal selection, and are found mostly in the 
splenic periareriolar sheath and intestinal lamina propria with a CD4/CD8 ratio of 2/1 . 
The last wave of thymocytes does not enter the thymus until El 8 and contributes 



38 

predominantly to the Vp2 + cq3 T cell subset, which makes up a very small percentage of 
the total T cell repertoire, develop like TCR2 cells, and are found in the spleen, but are 
rare in the intestine. They have a 4/1 ratio of CD4/CD8. Thus the three populations are 
produced sequentially in agreement with their ontogeny (Coltey et al., 1989). 

As in mammals, the chicken TCR genes in the chicken are structurally organized 
quite similarly to that of Ig chains and are evolutionarily conserved at the protein level to 
mammalian TCR. Key features of chicken TCR gene organization are shown in Figure 
1-8. The a and y chain variable regions are encoded by variable (V), joining (J), and 
constant (C) gene segments and join to form a sequence of V-J-C; the (3 and 5 chains are 
encoded by V, D, J, and C gene segments, with the D, or diversity, segments between V 
and J in order to form a sequence of V-D-J-C. The 8 chain locus is contained within the a 
chain locus. V, D, and J gene segments are flanked by typical recombination signal 
sequences (RSS) at the 3' end of V and D and the 5' end of D and J gene segments. As in 
the V region of the Ig molecule, there are three complementarity determining regions 
(CDR) and four framework regions (FR) in the V of the TCR molecule. These are 
involved in forming a stable three dimensional surface for binding antigen peptides 
presented in the major groove of the MHC molecule of an APC cell. The CDR3 is the 
most variable of the three CDR due to the high level of junctional diversity generated 
during TCR gene rearrangement. 

The chicken TCR-y locus has three Vy families, three Jy segments, and one Cy 
segment. Eight to ten members (with high homology) are found in each Vy family. The 
three Jy segments are more closely related to each other than to any in mammalian 



39 



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40 

species (Six et al., 1996), but most of the substitutions are in the silent second and third 
position in the amino acid code. TCRp locus includes two Vp families, one Dp, four Jp, 
and one Cp gene segment. There are approximately 6 members of the Vpi family and 
three to five of Vp2 gene segments. Within each Vp family there is little difference, 
however, the two families bear little similarity. 

So it appears that combinatorial rearrangement alone provides a somewhat limited 
means of generating diversity in chicken T cell receptors. No sequence modifications 
occur in the germline gene segments from recombination alone. In a sequence analysis of 
TCRp, Cooper and colleagues realized that diversity was generated almost exclusively in 
the junctions, creating nontemplated N regions. Every clone was found to have a distinct 
sequence at these N junctions between V-D and D-J in the CDR3 (Cooper et al., 1991). 

This is quite in contrast to humans and mice where there are about 50 functional 
V gene segments in 20-30 subfamilies of Vp, plus two separate clusters each consisting 
of a single Dp gene segment, a jp region (with 6-7 members each), and a single Cp gene 
segment. In humans there are eight Vy gene segments with five Jy segments arranged in 
two clusters and one Cy to yield 40 V-J pairings. In mice seven Vy and four Jy genes are 
arranged in four V-J-Cy clusters (Arden et al., 1995a, 1995b; Janeway and Travers, 1997; 
Rowenetal., 1996). 
T cell repertoire analysis 

Despite the fact that T cell repertoires may be as large as 10 15 specificities, it 
appears that most of the T cell responses studied in animal autoimmune diseases have 
demonstrated restricted repertoires of responsive clones, that of oligoclonal T cell 



41 

repertoires (Gold, 1 994). A more refined observation is that "most pathological infiltrates 
are either oligoclonal in nature or display oligoclonal expansions over a polyclonal 
background" (Pannetier et al., 1995). This is despite the fact that there may be a 
difference in length in the CDR3 found in any Vp-jp recombination junction of as many 
as 6-8 amino acids. For example, in experimental allergic encephalitis, T cells 
responding to the major epitope of amino acids 1-11 of myelin basic protein express Vp 
8.2 associated with either Vcc2 or Voc4 (Acha-Orbea et al., 1988; Urban et al., 1988). In 
rheumatoid arthritis, Palliard et al. (1991) and Howell et al. (1991) suggested that a 
superantigen activated the preferred T cells expressing Vp3, Vpi4, and Vpi7. Of these 
activated families, only T cells with specificity for synovial joint-associated antigens 
would then initiate autoimmune inflammatory response. These examples illustrate the 
limited subgroup of actual T cells clones (variants) being recruited, even though a vast 
number are available in the total repertoire. 

If that is so, then perhaps direct targeting and functional deletion of T cells that 
express specific V gene products can control the autoimmunity and still maintain an 
otherwise intact immune system. That would be like the elimination of certain T cells 
that respond to the minor lymphocyte stimulating antigen (Mis) for maleness in the 
mouse (Scott et al., 1995). Moreover, during the course of an autoimmune disease, 
evidence indicates that the expressed repertoire evolves, as more antigenic determinants, 
previously cryptic, become available and the immune response spreads to respond to 
them. What may happen in the EAE system, is that the original Ac 1 - 1 1 -specific T cells 
may upregulate self-antigen exposure in the CNS (a usually privileged site), activating a 



42 

newer set of T cells to what were until then nonsequestered cryptic MBP determinants 
(Lehmann et al., 1993). The reverse situation may occur in which the autoimmune 
response is diverse initially but honed via additional waves of recruitment, which may 
cause a consolidation and selection to produce a more oligoclonal T cell repertoire. So 
the conflicting reports between restricted or diverse repertoires may just represent 
different stages in the development of disease. 

Other chicken models of autoimmunity 

There are two other chicken animal models for autoimmune diseases in humans. 
The Obese Strain (OS) chicken line is characterized by iodine-induced autoimmune 
thyroiditis, and is recognized as a model for the organ-specific disease Hashimoto's 
thyroiditis. Reducing thyroidal iodine by antithyroid drugs can prevent the thyroiditis. 
However, therapy must be administered at the embryonic stage (Bagchi et al., 1995); 
otherwise, the thyroiditis becomes severe by 5 weeks of age. Autoreactive B and T cells 
can be seen in the thyroid by 2 weeks post hatch (Wick et al., 1970). Furthermore, 
adoptive transfer of splenocytes from affected OS chickens to the Cornell strain (CS), a 
related strain that develops a mild late onset disease, causes the development of 
thyroiditis when the hosts were supplemented with iodine (Brown et al., 1991). It has 
been recently demonstrated that the T cells expressing V(31 genes are the main T cells 
infiltrating the OS strain thyroids (Cihak et al., 1995). 

The University of California at Davis (UCD) lines 200 and 206 chickens develop 
a hereditary scleroderma-like connective tissue disease. It develops early in life, as early 
as 7 days post hatch, presenting initially as swelling, erythema, and necrosis of the comb, 



43 

digits, and skin (Haynes and Gershwin, 1983). Survivors develop a severe lymphocytic 
infiltrate of the comb, skin, digits, and viscera. They develop an excessive buildup of 
collagen, resulting in fibrosis of the dermis and as vascular occlusions of internal organs 
such as the esophagus, small intestine, lungs, kidneys, heart, and testes. T helper and T 
cytotoxic cells are present with a CD4:CD8 ratio of 1.44:1 by week four (van der Water 
et al., 1989). The infiltrates also contained distinct groups of B cells as the disease 
progressed. These infiltrates secrete IgM, fibroblast-activating cytokines (Duncan et al., 
1995), antinuclear antibodies (including antibodies to ssDNA), and anticytoplasmic 
antibodies that recognize an avian-specific set of antigenic determinants (Haynes and 
Gershwin, 1983). As in humans, fibroblast activation is suggested to contribute to fibrosis 
(Duncan et al., 1992). A defect in the T cells' response to a panel of T cell mitogens such 
as concanavalin A or pokeweed mitogen indicates abnormalities in T cell stimulation as 
seen in decreased calcium influx and proliferation (Wilson et al., 1992). 

Limitations in the use of the chicken animal model 

No animal can represent perfectly what is found in a human. Small mammals 
have become the more popular study models and often they can depict the human 
phenomenon reasonably well. In the case of vitiligo, the chicken presents a closer animal 
model due to the shared features suggesting an autoimmune component to the disease. 
However, the chicken is not well regarded as a relevant animal model for medical 
research, certainly not as extensively as the laboratory mouse. With a much more 
limited pool of investigators and experience, there is not an extensive network of shared 
technical protocols that have been developed or people aware of the chicken system to 



44 

begin trouble shooting this untapped resource. It takes time and a collective effort to try 
new ideas in the chicken just as it was for pioneers with the small mammals. Beyond not 
knowing if a protocol may work for a specific strain is whether the protocol already 
established in the mammal can be adapted to the chicken at all. 

It takes 5-6 months for chickens to become sexually mature so creating congenic 
chickens with 12 or so crosses would require several years. Slow reproduction is a 
problem with animals larger than mice. Larger animals are more costly to feed, house, 
and have enough space for. Because the chicken is not used extensively, researchers do 
not attempt new technology with them and the biotechnology industry does not find the 
need to develop useful tools. Transgenic chicken embryos are created with the assistance 
of infections by variants of the Rous Sarcoma Virus. Hybridomas have been developed 
only in the last few years. 

The genome of the chicken is just beginning to get serious attention and now only 
in the past 2 years has linkage mapping of the genome with readily available 
microsatellite markers is being started. Unlike the mouse, chickens have not been 
characterized genetically into well-defined lines guaranteeing the purity of a line. There is 
no equivalent for chicken of a library such as that of Jackson Laboratories that allows a 
scientist to buy a mouse, C57BL/6, or the NZW for its specific genetic features. The 
library of described avian cell differentiation antigens and known avian cytokines and 
lymphokines is also less extensive than that of the mouse and human. This limits the 
extent of some types of avian research, such as the cytokine profile expressed by cells in 
response to inflammation of the thyroid in the Obese Strain chicken. 



45 

A conclusion that can be made about using the chicken to study diseases in the 
human is that it will probably not be as readily appreciated as the laboratory mouse. It is 
more expensive in terms of reproduction time and cost to maintain. One really needs to 
be both a poultry and medical scientist. The typical medical scientist is not aware of the 
extensive network, knowledge, and experience that is necessary to study live chickens. 

On the other hand, the chicken is the best studied vertebrate for embryogenesis 
and development. The egg provides a most convenient source of embryos; the shell can 
simply be opened to expose the living animal. It was through studying the chicken, that 
the concept separating the B cells as bursa-derived (bone-marrow-derived for mammals) 
from the T cells, as thymus derived, was clarified. Several monoclonal antibodies 
including those that distinguish several subsets of T cells and B cells are available and 
marketed for cell separations and immunohistochemistry. At this moment, the Smyth 
line chicken does represent the closest model providing the best opportunity to unveil 
some of the unknown pathology of human vitiligo. 

Rationale for this study 

Vitiligo is considered an autoimmune disease. Vitiligo is not considered life 
threatening such as the physiological destruction of the insulin-producing cells in 
diabetes; nevertheless, for the 1-2% of the population with vitiligo there is a significant 
increased risk of skin cancer. The patient's well being and self esteem are compromised 
and these may cause distress because there is little the patient can do to hide the 
condition. The current means to treat vitiligo is to have the patient undergo PUVA 



46 

therapy to attempt to stimulate new melanization However the treatments are harsh, 
prolonged and the success rate is poor. 

Often the presence of one autoimmune condition can be found to occur along with 
other preexisting autoimmune conditions and this is true for vitiligo. For example, vitiligo 
has been associated with Autoimmune Polyglandular Syndrome type 1 and vitiligo 
patients have increased risk for other autoimmune diseases. 

Animal models have proven to be very useful in studying the pathogenesis of 
autoimmune diseases. In the study of diabetes mellitus, the target of autoimmune 
destruction is the P islet cells of the pancreas. The NOD mouse has been a very useful 
animal model to study the human disease. The insulitis has an infiltration mainly of CD8 + 
T cells but also of CD4 + T cells to a lesser degree. The disease has been proven by many 
labs to be adoptively transferred using T lymphocytes. Immunotherapy targeted at the T 
cell has been used inhibit the progression of the disease. Autoantibodies against the islet 
cells are characteristic of diabetes. They proceed and are detectable before the onset of 
the disease. GAD has been identified as a major autoantigen; both forms of which have 
homology with a peptide from Coxsackie virus. Islet cell antigen (ICA) has more recently 
been identified as a autoantigen in diabetes. Yet, the role of the autoantibodies in causing 
the destruction is not clear. 

The Smyth line chicken is the best available animal model for the study of human 
vitiligo. The melanocytes of the regenerating feather compare to the melanocytes of 
human epidermis and hair follicles. The depigmentation develops in patches that are 
irregular and expand as the process proceeds. The disease is otherwise asymptomatic in 



47 

both species, except in nearly 50% of the affected that become blind as well. There are 
similar inherent defects within the chicken melanocyte. An intense lymphocytic infiltrate 
with increased numbers of CD8 and CD4 T cells is associated in the chicken. 
Autoantibodies are detectable before the onset of amelanosis. A putative autoantigen, 
tyrosinase-related protein, has been detected and is related tyrosinase which is an enzyme 
involved in melanogenesis (Austin and Boissy, 1 995). 

Thus in this study of vitiligo, experiments were designed to ask some of the same 
questions as in other animal models of autoimmunity. Adoptive transfers of lymphocytes 
from affected SL chickens into non-affected BL chickens tested the hypothesis that 
autoimmune lymphocytes can induce the destruction of the feather melanocytes and 
cause the depigmentation. Likewise, the hypothesis that sera containing autoantibodies 
might induce disease if transferred to an unaffected host was also tested. 

Since T cells have been shown in diabetes to cause the disease, the question of 
which subsets of T cells might be the key members involved would help define the 
pathology. In the peripheral blood of Smyth line chickens, the y8 T cells increase in 
proportion during the course of the amelanosis and with age. Therefore, a repertoire 
analysis of the y8 T cells in the peripheral blood was examined. 

Since animal models allow one to manipulate the genotype of the animals to 
determine genetic causes of diseases, preliminary genetic studies of the SL chickens were 
performed. If certain patterns of inheritance are found that would correlate with the 
presence, absence, onset, or severity of the disease then this would help in the 
understanding and predictability of the disease. Endogenous viruses are genes that 



48 

encode components of retroviruses that have become integrated in the genomes of all 
species of vertebrate animals. They are stable and inherited in Mendelian fashion. The 
random integrations provide unique genetic markers that can be used to follow 
inheritance and examine for correlations with phenotype. 

It is a goal of using animal models to provide a means to gain understanding of a 
human disease. This study of the amelanosis in the Smyth line chicken is being pursued 
to understand vitiligo in humans. 



CHAPTER 2 
ADOPTIVE TRANSFER OF AMELANOSIS IN THE SMYTH LINE CHICKEN 



Introduction 



This aim is designed to determine the role and clarify the contribution of humoral 
and cellular immunity in the pathogenesis of amelanosis in the Smyth line chicken. 

In order to study and characterize the immune response, it is often an advantage to 
study the intact organism. To make the study manageable, experimental animals have 
been manipulated by various means to help study immune functions. The laboratory 
mouse for example, has been inbred so that the immune responses based on the MHC 
haplotype have been characterized and documented to minimize 'masking' of the effect 
of a locus or genetic region; a large collection of MHC-specific strains is available 
through Jackson Laboratories. This allows researchers to choose mouse strains to 
conduct investigations and make variants such as the NOD mouse. The variants have 
been developed through altering the genome either by inserting new genes to create 
transgenic animals, or by targeted disruption of genes by gene knockout through means of 
homologous recombination. 

Adoptive transfer of cells or antibodies is a classical experimental approach to 
demonstrate immune function. The transfer of serum (the fluid phase of blood containing 
specific antibodies against an immunizing antigen) from an immunized individual 
(donor) into a naive individual (host or recipient) can confer immunity if antibodies 

49 



50 

mediate that condition. This is passive immunization, dependent upon the antibodies 
generated originally by the donor by active immunization or infection. Adoptive transfer 
of lymphoid cells from the immune donor can provide cell-mediated immunity in a host. 
Transfer of cells must be done between donors and recipients genetically matched at the 
major histocompatibility complex (MHC) loci so that the donor cells are not rejected by 
the recipient and do not attack the recipient's tissues (graft versus host disease). 
Incompatibility may also occur despite the donor and host being identical at the MHC 
locus, due to differences in the minor histocompatibility antigens (Scott et al., 1995), such 
as the male specific H-Y antigen. 

Immunosuppression of the host animal is often utilized to facilitate adoptive 
transfer studies because syngeneic matches in the MHC are rare in outbred populations. 
This pretreatment also provides a void in the immune function in the recipient host 
providing space for the restoration of immune function by the adoptively transferred cells 
(Toivanen et al., 1975). This allows the effect of the transferred donor cells to be studied 
in the absence of host lymphoid cells. One method of immunosuppression is by the use 
of ionizing radiation from X-rays or y-rays to kill off rapidly dividing lymphoid cells at 
doses that spare the other tissues of the body. Other means of cell depletion include 
neonatal thymectomy, cyclophosphamide (which acts primarily by eliminating B cells 
and suppressor T cells) (Toivanen et al., 1975; Harada and Makino, 1984), splenectomy, 
and antilymphocyte antibodies generated in another species of animals. 

Adoptive transfer studies have been performed to study the functions of chicken 
lymphocyte subsets. Toivanen et al. (1975) compared the transplantation of lymphoid 



51 

cells (bursa, spleen, or bone marrow) into 4.5 week old immunodeficient chicks. Using 
donated bursal cells of 3 day old, 4.5 week old, or 10 week old donors, and pretreatment 
with cyclophosphamide (Cy), even large numbers of donated bursal cells would not bring 
about a long term restoration of antibody formation. Pretreatment by X-irradiation (750 
rads) on the day before transplantation with 10 week old donated cells from spleen (as 
well as marrow, thymus, or bursa) allowed higher survival rates and body weight gains 
suggesting that restoration of T cell functions, but not B cell functions, was achieved. 
Toivanen and colleagues concluded from these studies that T cell function is more crucial 
to survival than is humoral immunity. A dose of 750 rads for 4.5 week old chickens was 
shown to be effective in allowing reconstitution of the T cell compartment, but only short 
term reconstitution of B cells. 

Lehtonen and co-workers (1990) determined that Cy treatment destroys 
proliferating B cells in the bursa, and allows donor B cell reconstitution in 4 day old hosts 
for at least 10 weeks if 4 day old donor bursa cells were used. Irradiated with 750 rads, 4 
day old hosts could again be reconstituted with T cells but not with B cells. The B cell 
compartment was not restored. Based mainly on these reports, our experiments utilized 
irradiation treatment for the adoptive cell transfer of lymphocytes from amelanotic SL 
chickens into BL hosts, in order to determine whether the autoimmune disease could be 
transferred by lymphocytes. 

The transfer of autoimmune disease to host animals has been previously 
demonstrated by the adoptive transfer of lymphocytes in other animal models of 
autoimmunity. Experimental allergic encephalomyelitis (EAE) is a disease produced by 
injecting animals with homogenized spinal cord, myelin basic protein (MBP), and it 



52 

resembles the demyelinating disease similar to multiple sclerosis. It has been transferred 
using MBP-reactive T cells from the spleen or lymph node cells from a MBP-immunized 
donor to nai've syngeneic hosts in mice and rats (Panitch and McFarlin, 1977; van der 
Veen et al., 1989). In EAE, it appears that CD4 + T cells expressing a restricted, limited 
TCR repertoire are responsible. In humans, however, the TCR repertoire may be more 
diverse, with greater heterogeneity of MBP-specific T cells associated with a greater 
severity of disease (Richert et al., 1995; Utz and McFarland, 1994; Utz et al., 1994). 
Systemic lupus erythematosus was transferred to SCID mice when human PBMC were 
injected into SCID mice, and the SCID serum was shown to carry the human 
autoantibodies for up to 22 weeks (Ashany et al., 1992). 

In the non-obese diabetic (NOD) mouse, a model for human insulin-dependent 
diabetes mellitus, several laboratories have demonstrated the transfer of insulitis and 
diabetes into irradiated hosts. Normally, signs of initial insulitis begin to appear by the 
sixth week. By 30 weeks of age, spontaneous diabetes develops in about 95% of the 
mice with a mononuclear cellular infiltrate within the pancreatic islets (Wicker et al., 
1986). Wicker and coworkers induced diabetes within 3 weeks in greater than 95% of 
the hosts when the hosts were older than six weeks of age and by using unfractionated 
splenocytes from overtly diabetic NOD donor mice older than 1 6 weeks old (Wicker et 
al., 1986). They refined these studies further by achieving successful adoptive transfers 
using splenic T cells using the CD4 + or CD8 + T cell subsets (Miller et al., 1987). 
Meanwhile, Hanafusa and colleagues (1988) induced insulitis in T cell-depleted NOD 
mice reconstituted with the same two T cell subsets. Subpopulations of spleen and 
lymph node cells transferred diabetes to syngeneic neonates and demonstrated an age 



53 

and cell dose-dependent susceptibility range (Bendelac et al., 1987). LaFace and Peck 
(1989) transferred diabetes in non-susceptible C57BL/6 or B10.BR/cd mice, and Serreze 
and co-workers (1988) did the same in NOD SCID hosts. T cells have been generally 
recognized as being mediators of autimmunity against the pancreatic (3 cells in diabetes. 

Autoantibodies can also be transferred from autoantigen-immunized donors to 
induce the autoimmune disease in hosts. Autoantibodies to the thyroid stimulating 
hormone receptor from mothers with Grave's disease frequently produce thyroid 
activation when serum is transferred into the fetus. Because IgG can cross the placenta, 
infants of affected mothers can be born with hyperthyroidism (Gossage and Munro, 1985; 
Becks and Burrows, 1991). Thyroiditis enduring for up to 40 days has been induced by 
the transfer of antiserum to susceptible strains of mice (Tomazic and Rose, 1975). 
Likewise in the Obese Strain chicken, the chicken model for thyroiditis, repeated 
injections of high titer antiserum for 4 weeks induced thyroiditis (Jaroszewski et al., 
1988). In EAE, serum transfer in a rabbit model induced severe autoimmune thyroiditis 
(Inoue et al., 1993). Autoimmune cataract formation has been created experimentally in 
eyes of mice by means of serum and monoclonal antibody transfers from donors which 
had received injections of emulsified beta-crystallins (Singh et al., 1995). 

A current hypothesis of the pathogenesis in the Smyth Line chicken suggests that 
there are inherent defects in the melanocytes, predisposing SL melanocytes to abnormal 
antigen presentation (Smyth, 1989; Austin and Boissy, 1995; Sreekumar et al., 1996). 
Cells and antibodies in the SL chicken become sensitized to melanocyte antigens. 
Presumably auto-reactive T cells associated with the melanocytes can be seen infiltrating 



54 

the feather barb ridges in tissue sections stained with monoclonal antibodies specific for 
T cell markers (Erf et al., 1995b). Austin and colleagues reported the detection of 
melanocyte-specific antigens between 65 and 80 kDa in the Smyth line chicken (Austin et 
al., 1992). More recently, Austin and Boissy (1995) reported that these same 
autoantibodies are recognizing the chicken homologue of mammalian tyrosinase-related 
protein- 1 (TRP-1). 

Consideration must be given to the fact that during a long term condition as in 
autoimmunity changes will occur in the autoimmune repertoire during the course of a 
disease. Autoimmunity may represent not only the breakdown of self-tolerance, but the 
display of new cryptic self-determinants to which the host was not originally tolerant 
(Lehmann et al.; 1992 and 1993; Sercarz and Datta, 1994). The changes in the antigenic 
determinants that are involved in this amelanosis may therefore be reflected in a changing 
autoimmune T cell and B cell repertoire. The design of adoptive transfer experiments, 
might, therefore, take into account these possible shifts in antigenicity, and utilize donor 
cells from donors of different ages. 

The parental Massachusetts Brown Line chickens (BL) from which the Smyth line 
was derived also exhibit the amelanosis of the feather and eyes but at an incidence of only 
1 to 2 percent (Erf et al., 1995a) as compared to the 90% incidence reported for SL 
(Smyth, 1989; Smyth et al., 1981). One potential explanation for this low incidence of 
amelanosis might be that the melanocytes of some BL chickens display the same defects 
in antigen presentation as hypothesized for the SL. The adoptive transfer of autoreactive 
lymphocytes from amelanotic SL chickens may result in the same autoimmune 
pathogenesis. Alternatively, the melanocyte defect may not be required, and the simple 



55 

presence of anti-melanocyte autoreactive lymphocytes, or autoantibodies, may be 
sufficient to cause amelanosis in BL chickens receiving SL lymphocytes or serum 
autoantibodies by adoptive transfer. The variables tested included the host age, donor 
age, use of irradiation, and number of injections of donor lymphocytes. We also 
performed one experiment involving the transfer of SL serum autoantibody into BL hosts. 
In this study, we report the first transfer of autoimmune amelanosis with splenic cells 
from Smyth Line chickens in Brown Line chickens. 

Materials and Methods 

Animals 

Fertile SL eggs from the B^°l major histocompatibility complex (MHC)-defined 
subline, which has the earliest age of onset and the most severe phenotype of the three SL 
MHC-defined sublines (Erf et al., 1995a), and from the B 101 MHC-matched BL were 
generously provided by Dr. J. Robert Smyth, Jr. (University of Massachusetts, Amherst). 
Chickens were hatched and housed at the University of Florida Poultry Unit, and were 
individually identified with leg band or wing tag numbers. The degree of pigment loss 
(amelanosis) by SL chickens was classified according to Erf et al. (1995b): (1) normal, 
no apparent amelanosis; (2) mixed amelanosis, with both normal and <20% amelanotic 
feather tissue; (3) mixed amelanosis, with normal and 20-60% amelanotic feather tissue; 
(4) mixed amelanosis, with normal and >60% amelanotic feather tissue; and (5) complete 
amelanosis, all developing feathering tissue is amelanotic. SL chickens with stage 1 or 
no amelanosis are also referred to as nonprogressors, and SL chickens with any apparent 






56 

amelanosis (stages 2-5) are referred to as progressors. It should be noted that the 
phenotypes reported represent the maximum amelanosis stage reached and were stable. 

The SL donors were hatched and raised before hatching the BL hosts so that there 
were visible amelanotic donors by the time they were 8-12 weeks old in time to donate 
their cells to 3 week old BL hosts. 
Sex Determination by PCR 

The sex of the donors and the recipients was determined when the hosts were 3 
weeks old because the hosts at this age do not have definitive secondary sexual features 
for reliable identification. SL females display the earlier onset and more severe phenotype 
than SL males and would appear to have the best potential of passing autoreactive T cells. 
The ideal transfer would be from a SL female to a BL female. Attempts were made in 
some experiments to avoid female to male cell transfers in case of possible 
incompatibility at the minor H loci. In birds, the females are the heterogametic sex, 
bearing the W and Z chromosomes whereas males are ZZ. The gender was determined 
by PCR using primers, Chi I and Chi III, derived from the sequence of the chicken W 
chromosome-specific Xho repeat fragment described by Kodama et al. (1987) and kindly 
provided by Dr. Siwo R. de Kloet (Florida State University, Tallahassee). The DNA 
template for the PCR was obtained from a drop of blood obtained by pricking the brachial 
wing vein and absorption onto Isocode Stix, PCR template preparation dipsticks 
(Schleicher and Schuell Inc., Keene, NH). Genomic DNA was eluted and PCR amplified 
as directed by the manufacturer. A 316 bp product was resolved on a 1% agarose gel. 



57 

Immunosuppression of the Host Animals 

Some host BL chicks were immunosuppressed by sublethal irradiation one day 
before the cell transfers, y irradiation was performed using a 137 Cs source at the U.F. 
Health Center Animal Resources Facility. Dosages used were either 750 rads (Toivanen 
et al., 1975, as used on 4 day old chicks) to 850 rads (Dr. Bruce Glick, personal 
communication) or none at all. 
Preparation of the SL Donor Cells and Cell Injections 

The donor SL cells were obtained from SL chickens undergoing active amelanosis 
at the time of sacrifice (8 to 20 weeks of age). The spleens were removed, made into cell 
suspensions in PBS on ice by teasing the organ and by dounce homogenization, and the 
lymphocytes were separated from the red blood cells by density centrifugation over 
Ficoll-Hypaque (Pharmacia). The splenic lymphocytes were resuspended in PBS at a 
density of 5xl0 7 to lxlO 9 cells per ml, and a maximum of 1-2 ml of cell suspension was 
then injected intravenously into the right jugular vein with the balance into the wing 
brachial vein. The recipient animals were then monitored in normal housing conditions 
(not pathogen-free) for at least 20 weeks of age to allow the development of the 
amelanotic phenotype during the typical time frame as would be found in a SL chicken. 
Smyth Line Serum Collection and Preparation 

Serum was collected from Smyth Line chickens that displayed obvious 
amelanosis (at least level 3) at the time of collection. A range of 20 to 60 ml of blood per 
bird was collected into heparinized Vacutainer tubes from the jugular and/or brachial 
vein of the wing. Blood was centrifuged at 1500 rpm to collect the serum, which was 






58 

then stored at -80°C. The sera was then pooled and the gamma globulin fraction was 
obtained by two sequential precipitations with 33% and then 28% saturated ammonium 
sulfate. The precipitates were dissolved in a minimum volume of cold phosphate 
buffered saline (PBS), dialyzed 2-3 days against 4°C PBS after each precipitation, and 
then filter-sterilized using a A5[i micropore filter (Nalgene). Aliquots of the gamma 
globulin fractions were then introduced by injection in the jugular vein (1-2 ml) with an 
additional volume (2-3) introduced intraperitoneally beneath the breastplate. 
Cell Lines 

Chicken melanocyte cultures were obtained from chicken embryonic neural crest 
tissue by the method of Boissy and Hallaban (1985) in the laboratory of Gisela Erf and 
obtained as a gift. Cultures were grown in Ham's F10 media (Sigma) supplemented with 
10% FBS, 5% Nuserum (Collaborative Biomedical Products), 200 mM L- 
glutamine/penicillin-streptomycin (Sigma), 0.5 mg/ml cholera toxin (Sigma), and ImM 
phorbol myristic acid. 
Immunoblotting 

Semiconfluent melanocytes were harvested from flasks, rinsed twice in PBS and 
solubilized in 10 mmol/L Tris buffer (pH 8), with 1 mmol/1 phenylmethysulfonyl 
fluoride, 5% 2-mercaptoethanol, .02 mM/1 of each antipain, aprotinin, chymostatin, 
leupeptin, and pepstatin A, with 1% sodium dodecyl sulfate (SDS) for 5 minutes at 95- 
100° C, and sheared with 21 GA needle. The lysates were separated on SDS- 
polyacrylamide gel electrophoresis (SDS-PAGE) reducing gels and electroblotted onto 
Immobilon-P polyvinylidene fluoride (PVDF) membrane. A Bio-Rad low range 



59 

molecular weight marker lane was used (Bio-Rad Laboratories, California). The blotted 
proteins were blocked in 5% Carnation non-fat dry milk with 20 mM Tris pH 7.5, 137 
mM NaCl, and 0.1% Tween 20 (TBS-T). The blots were cut in individual strips and each 
strip was individually incubated with different sources, primary antibody (either direct 
serum cleared as described above or the gamma globulin fraction) for 2 hours at room 
temperature in TBS-T with 5% milk, washed 5 times for 2 minutes, and 2 times for 5 
minutes and then incubated in goat anti-chicken horse radish peroxidase-conjugated 
secondary antibody (Southern Biotechnologies, 1:1500) for 2 hours. The proteins were 
detected by ECL chemiluminescence (Amersham). The blots were analyzed by 
densitometry and Hoefer Image Master software. 
Histology 

Regenerating feathers of SL and control BL birds were generated by gentle 
plucking of growing feathers and collecting of the young feathers bimonthly. The 
feathers were brought to the University of Florida Diagnostic Research Laboratory for 
cryosectioning. Mouse monoclonal antibodies to chicken TCR1, TCR2, TCR3, CD4, 
CD8, CT3 were a gift of Dr. Chen-lo Chen and described by Cooper et. al., 1991). 
Methyl green was used as the counterstain. 

Results 

Observations of the UF Colony of Smyth Line Chickens 

Observations have been compiled from raising twelve populations of the Smyth line 
chicken. In the colony raised at the University of Florida, the amelanosis incidence has 
been approximately 60%. Observations compiled from four of the populations of Smyth 



60 

line chicken is summarized in Table 2-1. Female Smyth chickens (Table 2-1 and Figure 
2-1) usually demonstrate a quicker onset and more severe manifestations of the 
phenotype of depigmentation at a higher frequency than in males, a phenomenon that has 
been seen in other autoimmune disease animals such as the female NOD mouse. 

During the first 6-9 weeks, 42% of the females (16 of 38) became amelanotic, 
with 12 at the more severe levels (stages 3-5). As the birds aged, the females continued to 
have a higher proportion that are affected and have the more severe phenotype. By the 
17 th week, 66% of the females were amelanotic and of these, 20 displayed the more 



Table 2-1. Amelanosis incidence in the UF Smyth line colony 



Population 


# number 
available 


early onset 
(6-12 wks) 


delayed 

onset 

(17 wks) 


number 
amelanotic 


% 
amelanotic 


total 
amelanotic 


SL2 


6F 


5/6 


1/6 


6/6 


100% 


60% 




11M 


3/11 


1/11 


4/11 


36% 


10/17 
















SL3 


14F 


9/14 


1/14 


10/14 


71% 


77% 




8M 


7/8 





7/8 


87.50% 


17/22 
















SL11 


9F 


4/9 


3/9 


7/9 


77% 


68% 




16M 


8/16 


1/16 


9/16 


56% 


17/25 
















SL12 


17F 


5/17 


2/17 


7/17 


41% 


36% 




11M 


2/11 


1/11 


3/11 


27% 


10/28 
























total F 


72% 


60% 










total M 


52% 





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64 

severe phenotypes. Only 14% of the males (4 of 28) showed any depigmentation by the 
9 th week and all at the lowest level (Table 2-1 and Figure 2-2). Males can become just as 
severe; they usually take longer to develop. By the 17 th week, 42% displayed the more 
severe phenotypes, but only one showed the severest at level 5, as compared to 9 females. 
The information combining the frequencies from both sexes is compiled in Figure 2-3. In 
one generation, there was actually some reversion of the phenotype in three of the 
females, otherwise the phenotype once the maximum was achieved remains stable. Of 
these three revertants two were completely amelanotic and one was 75% amelanotic. 
After their initial depigmentation had peaked, a period of repigmentation began, which 
started as patches in a different pattern than they were first depigmenting. The 
repigmentation was not complete. 

There seems to be two waves of developing amelanosis based on observations of 
several populations as shown in Table 2-1 and Figure 2-3. The early onset wave 
(majority of the SL) begins at the 6 th through the 9 th week post hatch. They peaked at 
development of amelanosis by the 12 th to 14 th week, achieving the severe phenotypes, 
stages 4 and 5, often earlier than 12 weeks. The second wave, fully pigmented up to this 
point, started developing amelanosis at 17 th to 20-2 1 st week and none became totally 
amelanotic from the second wave. This was seen at week 17 for a male from the SL2 
population and for two females from the SL 1 2 population. 
Adoptive Cell Transfer Experiments 

Five cell transfer experiments were conducted. Variables considered included: (1) 
host age; (2) donor age; (3) stage of amelanosis exhibited by the donor (including how 



65 

rapid and severe the amelanosis developed); (4) whether hosts were irradiated; and (5) the 
number of injections of cells. 

The first cell transfer experiment was performed on 18-19 day-old BL chicks 
(mean weight of 126 g). In order to allow for the possibility that changes might occur in 
the autoimmune T cell repertoire during the course of disease, two age groups of SL 
donors were used. The plan included five test groups with a one time donation of 
transferred cells per host, as shown in Table 2-2 (Groups 1A-1E). 

Total body irradiation of 750 rads was issued per host BL bird on the day before 
the adoptive transfer. The splenic lymphocyte suspensions were prepared with a range of 
cells between 5xl0 7 cells/ml and 4xl0 8 cells/ml, which is within the range used in 
mouse and rat transfer experiments. Hosts and controls were kept in normal housing 
conditions. Of the 25 BL host chickens, 12 survived. Of these 12 BL hosts that received 
SL donor cells, 5 (44%) displayed a partial amelanotic phenotype. This has been 
summarized in Table 2-3, which shows the progression of amelanosis of these 5 hosts 
after receiving the transfer of SL lymphocytes. 

Two females, from either groups 1A, ID, or IE, each developed a highly severe 
stage of amelanosis within 3 months of age. Unfortunately, these two died and were 
removed from the poultry unit by the animal caretaker before photographs and tissue 
samples could be taken. Unfortunate too, was the fact that the birds had outgrown their 
leg tags. Other BL hosts only hinted of a possible amelanotic phenotype. BL5-105, a 
female also from either groups 1A, ID, or IE, gradually developed amelanosis to stage 3 
in severity. Unfortunately, this healthy bird died suddenly before photographs were 
considered. It too was removed from the poultry unit before samples could be taken. 



66 



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67 



Table 2-3. Progression of amelanosis in 5 BL5 hosts after adoptive transfers of SL 
splenic lymphocytes. 



Age of host in months 


Animal 


sex 


3m 


4m 


5m 


6m 


7m 


8m 


9m 


BL5 


F 


4(a) 














BL5 


F 


4(a) 














BL5-105 


F 


1 


2 


3(b) 










BL5-111 


M 


1 


2 


2 


3 


3 


3 


3(c) 


BL5-115 


M 


1 


2 


2 


2 


2 


2 


2(c) 



(a) Died at 1 2 weeks old and removed from poultry unit before samples could be taken. 

(b) Died within the fifth month of age and removed before photographs and samples 
could be taken. 

(c) Died after the ninth month of age. 

There are five levels to describe the degree of amelanosis as developed by Erf et 
al. (1995b) and adapted by this lab: 

(1) normal, no apparent amelanosis; (2) mixed amelanosis, with both normal and 
<20% amelanotic feather tissue; (3) mixed amelanosis, with normal and 20-60% 
amelanotic feather tissue; (4) mixed amelanosis, with normal and >60% amelanotic 
feather tissue; and (5) complete amelanosis, all developing feathering tissue is 
amelanotic. SL chickens with stage 1 or no amelanosis are also referred to as 
nonprogressors, and SL chickens with any apparent amelanosis (stages 2-5) are referred 
to as progressors. 



Two males, BL5-111 and BL5-115, also developed an amelanotic phenotype 
resembling that of the Smyth line and were followed more closely. Around the sixth 
month of BL5-1 11 's life, regenerating feathers began to display melanocyte destruction 
in the pulps that had been all along reflected in the banded black and white feather vanes. 
Now the pulps in regenerating feathers were creamy gray instead of homogenous black. 
By the eighth month, the pulps in the regenerating feathers in BL5-1 1 1 were mostly gray, 
some were banded black and white, and only one to two percent were still completely 
black. B15-115, even in its eighth month, still displayed a low level of amelanosis with 
mostly black pulps in the regenerating feathers. Nevertheless, the feather vanes were 
banded with the oldest parts, the tips, being black and the youngest parts, closest to the 
pulp, being white or depigmented. The development of amelanosis was very gradual and 
not convincing until changes in the pulps could be witnessed. Photographs of the extent 
of amelanosis developed at the age of 7 months are shown in Figure 2-4 and 2-5. 

In the second cell transfer experiment, three groups of 12 day-old BL7 generation 
hosts (sex not predetermined) were used as the recipients of a single transfer of cells 
comparing hosts receiving lymphocytes from a SL donor to hosts receiving lymphocytes 
from a BL control donor (Table 2-2). 

Hosts were irradiated with 850 rad total irradiation each on the day before the 
adoptive transfer and given a single reconstitution of SL splenic lymphocytes or BL 
lymphocytes. An extra 12 BL7 which were prepared by irradiation were not used and 
never reconstituted with cells. These 12 survived until sacrificed after 20 plus weeks of 
age. 



69 




Figure 2-4. BL5-1 1 1, a Brown Line adoptive transfer host displaying stage 3 amelanosis 




Figure 2-5. BL5-1 15, a Brown Line adoptive transfer host displaying stage 2 amelanosis 



70 



Only three BL7 hosts showed any manifestations of amelanosis above, BL7-222, 
(male), showed amelanosis of stage 2. Out of 4 host BL chickens, reconstituted with SL 
donor cells, only one (25%) demonstrated amelanosis. None of the control hosts 
reconstituted with BL donor cells developed any signs of amelanosis. 

In the third adoptive cell transfer experiment, older 6 week-old non-irradiated, 
normal hosts were given one injection of SL donor splenocytes, as shown in Table 2-2 
(Groups 3A-3C). Donor cells came from two different age groups. The change to 6 
week-old chickens and no irradiation provided the opportunity of the donated SL splenic 
lymphocytes to be present during the time period in which amelanosis usually starts in 
the early onset SL chickens. None of the 10 host BL chickens developed any signs of 
amelanosis by the age of 20 weeks. 

In the fourth cell transfer experiment, a serial adoptive cell transfer protocol was 
adopted. Each set of BL hosts, which were not irradiated, received several cell injections 
from a series of amelanotic SL donors over a course of several weeks. The transfers 
began at 6 weeks of age (just before the time when amelanosis begins to appear in the 
SL) and continued through the time period (8-16 weeks) during which amelanosis usually 
develops in the SL (Table 2-4, Group 4). It was hoped that weekly repeated injections of 
splenic cells from SL donors into the same set of BL hosts would mimic the constant 
presence of melanocyte-sensitized T cells in the SL chicken. 

In experiment 4, 5 female BL7 hosts initially received SL donor cells representing 
three different experimental test groups, Groups 4A-4C. However, it was necessary to 
simplify the source of donor cells in the subsequent transfer of SL lymphocytes to one 



71 



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72 

source of donors each week. This was dictated by the availability of a large enough pool 
of SL donors with the desired stage of amelanosis and the work necessary to prepare the 
splenocytes from several SL donors. Thus if SL males provided the desired amelanotic 
features, their spleens were used to augment the SL donor cell source. The hosts in 
Group 4 were retained until 20 weeks of age; however, no amelanosis was apparent. 

The fifth adoptive cell transfer experiment also utilized 6 week-old nonirradiated 
normal BL hosts with the same protocol as in the fourth experiment. Seven BL9 female 
hosts were given injections of SL donor splenic lymphocytes on week 6 and then on week 
7 (Group 5A). A control group of 3 BL9 females received injections on both weeks of 
BL splenic lymphocytes (Table 2-4, Group 5B). These hosts were maintained for 18 
weeks, and no amelanosis was observed. 
Serum Transfer Experiment 

We performed one experiment to determine whether the transfer of SL serum 
autoantibodies to melanocyte antigens could result in the transfer of the disease into BL 
chickens as evidenced by an amelanotic phenotype. Repetitive administrations of serum 
have been utilized in previous studies of passive transfer of autoimmunity. For example, 
in the autoimmune murine model for thyroiditis, serum transfers were performed on day 
0, 2, and 4. This was sufficient to induce thyroid lesions by passive transfer of immune 
serum in 10-12 week old animals (Tomazic and Rose, 1975). The regiment of weekly 1- 
ml injections has been performed for 4 weeks by intravenous and subcutaneous means in 
the OS chicken model of thyroiditis (Jaroszewski et al., 1978). Serum was collected from 
SL chickens that expressed amelanotic phenotypes of various different stages (stages 2-5) 



73 

to account for the possible changes that occur in the antibody repertoire as amelanosis 
progresses. We collected and pooled serum from four different sources: 

Pool 1 = 10-11 weeks old (SL1 1) 

Pool 2 = 3-7 months old (SL6, SL7, SL10) 

Pool 3 = 11 months old (SL3) 

Pool 4 =17 months old (SL2) 

Eight normal BL10 hosts received SL gamma globulin injections over a 4-week 
period. Three BL10 hosts received PBS as a control group. 

A Bio-Rad protein assay was used to determined the total gamma globulin 
fraction administered per injection by assaying aliquots saved from each preparation used 
for injection. Results of this assay, (Table 2-5) indicate that the hosts received a total of 
99 to 121 mg of total serum gamma globulins, which corresponds to the original serum 
volumes of 82 to 116 ml per host. 

The experimental approach for this experiment combined elements of the 
aforementioned mouse (Tomazic and Rose, 1975) and OS chicken (Jaroszewski et al., 
1978) studies. It included 3 administrations during the first week and then additional 
inoculations weekly for four additional weeks. Each autoantibody (gamma globulin) 
transfer consisted of the injection of a total of 5 ml per host: 2 or 3 ml into the jugular 
vein and 2-3 ml injected intraperitoneally (Table 2-6). Hosts and controls were kept in 
normal housing conditions. 

These gamma globulin transfer hosts were maintained and monitored biweekly 
for visual changes in phenotype and regenerating feathers were collected biweekly. No 






74 



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75 















Table 2-6. Adoptive transfer of SL gamma globulin into 6 week-old BL10 hosts 



Day of 








Serum 




Injection 


Age 


Pool 


ml 


Equiv.(ml) 


mglgG 


1 


6 w 


2 


5 ml 


14 


10.7 


3 


6 w 


2 


5 ml 


14 


10.7 


6 


6 w 


2 


5 ml 


14 


10.7 


15 


7 w 


3 


5 ml 


11 


18.4 


23 


8w 


1 


1.5 ml 


6 


11.9 


30 


9w 


3 


5 ml 


11 


18.4 






4 


5 ml 


28 


28.9 


37 


lOw 


3 


5 ml 


11 


18.4 






4 


5 ml 


28 


28.9 



76 

induced amelanosis was apparent by the age of 1 8 weeks at which time it was decided to 
terminate the experiment, 8 weeks after the last injection of gamma globulins. 
Western Blot Analysis 

Western blot analysis was performed to confirm the presence of antimelanocyte 
autoantibodies in the serum pools used for the gamma globulin adoptive transfer 
experiments. Serum from the two adoptive cell transfer BL hosts that became melanotic, 
BL5-111 and BL5-115, were also examined to learn more about the extent of their 
phenotype. 

As shown in Figure 2-6, serum pools 1, 3, and 4 (lanes 1, 2, and 3 respectively) 
did contain autoantibodies specific to melanocyte proteins in the size range of 65-80kDa, 
reported as characteristic of the Smyth line chickens by Austin et al. (1992). Lanes 4, 5, 
6, and 7 are serum samples from BL adoptive cell transfer hosts, BL5-1 1 1 and BL5-1 15, 
from two timepoints each at about a month apart. All four samples had the same 
autoantibody profiles specific for the same melanocyte proteins as seen typical in the 
other SL sera (lanes 1, 2, and 3). This suggests that, in addition to the partially amelanotic 
phenotype observed in these hosts, antimelanocyte autoantibody production may have 
been induced by the adoptive transfer of SL splenic lymphocytes. Control sera included 
an amelanotic SL positive control that recognized the same 65-80 kDa protein bands, and 
a BL serum negative control (lane 8), which did not contain autoantibodies that would 
recognize the same melanocyte proteins. 

According to Austin and colleagues, the SL antimelanocyte antibodies recognize 
melanocyte proteins between 65 kDa and 80 kDa in size. Using Image Maker software, 



77 




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the principal bands observed between the 49.5 and 80 kDa molecular weight markers 
have a size of 65 to 80 kDa proteins (Figure 2-6). The 64-65 kDa protein band is seen 
across all lanes (see the lower of 2 bands next to 80 kDa marker) including in the BL 
(lane 8). 

A 79 kDa protein band was recognized by all the SL samples (lanes 1 -7) but not 
by BL samples, which corresponds to a protein reported by Austin and colleagues as SL- 
specific (1992 and 1995). This figure is representative of the results of three blots using 3 
different BL animals. None of the three BL control sera tested had antibodies that 
recognized the 79 kDa band. Austin and Boissy (1995) demonstrate that the melanocyte- 
specific proteins of the 65-80 kDa range are Trp-1 specific. This 79 kDa protein may 
therefore be Trp-1 . 

Regenerating feathers were plucked biweekly and collected of BL5-1 1 1 and BL5- 
1 1 5 starting from the fifth month. Cryosections were prepared and immunostained for 
CD3 expression using mouse monoclonal antibody as referenced in Chapter 1 (Erf et al. 
1995b; Cooper et al., 1991). As compared to feathers from a BL control chicken, the 
barb ridges of feathers from BL5-111 contained a gradually decreasing amount of 
melanin in the barb ridges (data not shown). The last sample taken early in its last month, 
was completely without melanin. This suggests that in the BL5-1 1 1 host, antimelanocyte 
lymphocytes had penetrated beyond the confines of the pulp and invaded the barb ridges 
to destroy and remove the melanocytes and their products. BL5-115 samples did not 
show these changes and resembled the fully pigmented barb ridges of the BL control. 
The tissue samples were poorly preserved due to improper freezing and so can not be 
published. These findings, in addition to the obvious change in phenotype and the serum 



79 

antibody profile, suggest several samples of evidence of the changes induced by the 
adoptive transfer of SL lymphocytes in this BL5-1 1 1 host. 

Discussion 

Adoptive transfer experiments often can provide a means to study the 
autoimmune response in vivo in the intact organism. We used this experimental approach 
to determine whether melanocyte-sensitized lymphocytes and/or autoantibodies have the 
capacity to transfer amelanosis in a non-Smyth line chicken. 

In hindsight, this portion of my project presented numerous technical challenges 
for a chicken animal model, including a relative lack of published protocols for adoptive 
transfer, radiation doses, etc. Most literature reported protocols in which mice were 
suppressed soon after sexual maturity (8-10 weeks). However, chickens are not sexually 
mature until the fifth or sixth month and that is unsuitable for the study of amelanosis in 
the SL. Most literature reported using approximately 5xl0 7 donated cells whether the 
cells were bone marrow cells, spleen, or lymph node cells. Our experiments involved the 
transfer of up to lxlO 9 cells. 

I had consulted several authorities on the chicken animal model, but never found a 
standard protocol for the immunosuppression of hosts of cell transfer that answered such 
questions as to its necessity, by which means, and how much per age or weight or other 
measurable parameter. Most published mouse and rat experiments did use irradiation 
(Wicker et al., 1986; Hanafusa et al., 1988; Panitch and McFarlin, 1977; van der Veen et 
al., 1989; Toivanen et al., 1975). Miller et al. (1988) subjected 4 to 8 week-old NOD 
mouse recipients with up to 950 rad of irradiation. However, within 2 hours the hosts 



were reconstituted with splenocytes. In our experiments, the irradiation occurred the day 
before. Irradiation was probably necessary based on the fact that the animals in our 
experiments that did display induced amelanosis had been irradiated. Like et al. (1985), 
working on the adoptive transfer of diabetes in the biobreeding rat, concluded that an 
"...an intact immune system protects against adoptive transfer and diabetes ...", and so 
they suggested the requirement for immunosuppression. An alternative to irradiation that 
could have been used is cyclophosphamide (Cy) which has been used to immunosuppress 
the hosts in the mouse and rat (Hanafusa et al., 1988) and in the chicken (Toivanen et al., 
1990; Lehtonen et al., 1975; Glick by personal communication and Glick, 1977; Olah and 
Glick, 1978; Glick, 1971). Transferring unfractionated splenic lymphoid cells produced 
higher incidence of insulitis than T cell subsets in the mouse (Hanafusa et al., 1988). 

A logical next step in our experiments would be to transfer T cell subsets. As 
demonstrated in various studies of the subsets of T cells mediating diabetes in the NOD 
mouse, some researchers report the both the CD4 + and CD8 + T cell subsets were 
necessary to transfer diabetes into recipients (Miller et al., 1988; Bendelac et al., 1987). 
Mitsunobu et al. (1992) concluded that CD4 + T cells cause the insulitis and that the CD8 + 
T cells act as mature killer cells against the P cells with the aid of CD4 + T cells. This 
ability to distinguish separate roles for different subsets of cells as demonstrated in the 
mouse should be possible in the SL chicken model for amelanosis. 

Mouse monoclonal antibody reagents specific for T cells expressing y8 (TCR1), 
apVpl (TCR2) and apvp2 (TCR3) (Chen et al., 1988; Chen et al., 1989; Char et al., 
1990) are available. The chicken homologues to CD3, CD4, CD8, CD1, CD2, CD5, 



81 

CD45, MHC class I, MHC class II, and IL-2 receptor are also recognized by mouse 
monoclonal antibodies (Cooper et al., 1991). Negative selection by means of complement 
lysis of the non-desired T cell subsets, or positive selection using mouse anti-TCR mAb 
and goat anti-mouse conjugated to magnetic beads are established methods. Another 
possibility would require the isolation of clonal populations from regenerating feathers 
using Con-A and IL-2 stimulated T cell subsets (Koevary et al., 1983; Panitch and 
McFarlin, 1977). This would most closely resemble the T cells at the site of melanocyte 
destruction (Erf et al., 1995b). There is the issue of the unknown feasibility of adopting 
this technique to the chicken in vivo. 

Nevertheless, the fact that 5 out of 12 BL5 hosts (44%) and 1 out of 4 BL7 hosts 
(25%) survived long enough to manifest amelanosis by adoptive cell transfer indicates 
that the amelanosis found in the SL chickens may be cell-mediated. A total of 40 BL 
hosts of SL splenic lymphocytes were followed from all five experiments and of these 6 
hosts (15%) became amelanotic. This is not by chance considering BL chickens normally 
demonstrate only 1-2% comparatively. These results suggest that the autoreactive 
lymphocytes are capable of recognizing BL, as well as SL, melanocytes, and that the SL 
melanocyte defect may not be required for pathogenesis. 

The T cell-mediated melanocyte destruction in vitiligo would resemble the T cell- 
mediated destruction of pancreatic p cells in diabetes, demyelinization in myelin basic 
protein-induced EAE, and destruction of follicular epithelial cells in autoimmune 
thyroiditis. T cell involvement has also been suggested in two other autoimmune chicken 
conditions, hereditary scleroderma in the UCD-200 and UCD-206 lines (Haynes and 



82 

Gershwin, 1983; van der Water et al., 1989) and thyroiditis found in the Obese strain 
(OS) chicken (Brown et al., 1991; Wick et al., 1970). The observation that adoptive 
transfer of SL lymphocytes may cause amelanosis in BL hosts is significant, in part 
because previous authors on the amelanosis of the SL chicken have over-emphasized the 
role of autoantibody, based on studies of bursectomy and corticosteroid inhibition of 
amelanosis (Lamont and Smyth, 1981; Boyle et al., 1987). 

It is also interesting to note that the western blot analysis demonstrated that the 
BL adoptive cell hosts had the same antimelanocyte serum antibody profile as the typical 
SL serum. This result suggests that either: (1) melanocyte-reactive SL B cells were 
transferred; or (2) melanocyte-reactive SL T helper cells induced BL B cells to produce 
anti-melanocyte autoantibodies. 

One must consider the possibility that the BL hosts that became amelanotic as a 
result of the cell transfers may actually have been susceptible to amelanosis given the 1 to 
2% incidence of amelanosis in the BL. The introduction of autoreactive SL lymphocytes 
may have "tipped the scale" in favor of disease in these individuals. This is the 
conclusion that adoptive transfer of splenocytes in the NOD mice accomplished in the 
work by Wicker et al. (1986). 

The repetitive administrations to the BL hosts with the transfer of SL-sensitized T 
cells or autoantibodies during the time period during which amelanosis has normally 
occurred in the SL donors appeared to be a logical approach to inducing amelanosis and 
should be done in combination with immunosuppression. Future studies should include 
these two conditions. Recall that in the first experiment the BL5 hosts were immune 
suppressed and were given one transfer of cells, resulting in five BL5 recipients that 



83 

displayed the amelanotic phenotype to some degree. The serial transfer experiments (3 
and 4) with older (6-week-old) not-immune-compromised hosts were given up to 5 
repeated transfers of donated SL cells. Still they had not displayed a hint of amelanosis 
in a situation we anticipated would have an earlier onset. Future experiments should 
involve both immunosuppression by irradiation and serial transfers. 

Previous attempts to induce an autoimmune disease by injection of antisera in 
normal chickens have been unsuccessful (Jaroszewski et al., 1978). Anti-thyroglobulin 
antisera from OS chicken suffering from thyroiditis were transferred to the normal 
Cornell strain but failed to induce thyroiditis, despite the fact that they share the same B 
alleles at the MHC locus. Immunocompromising these Cornell recipients did not help, 
even though neonatal thymectomy did potentiate the severity of thyroiditis in OS 
chickens (Wick et al., 1970), as immunosuppression seemed to have done for our cell 
transfer experiments. 

Timing of the transfers may have an influence on the success. Wicker et al. 
(1986) noticed that they were unable to transfer diabetes using splenocytes if the NOD 
irradiated mice were less than or equal to 6 weeks old, but were much more effective with 
transfers to hosts that were slightly older than 6 weeks of age. For the Smyth line 
chicken, perhaps the transfers needed to be initiated earlier for both our cell and humoral 
administrations. Perhaps in vitro stimulation of the splenocytes with lectins or 
melanocyte fragments in the donor may help (Takenaka et al., 1986; McCarron and 
McFarlin, 1988). Cell transfers have been successful from bone marrow and mature 
splenocytes or lymph node cells. Our utilization of repeated transfers of cells or gamma 
globulin, and the concentration of the immunoglobulins used should have addressed the 



84 

problem of maintaining high enough concentrations of effector cells or antibodies over 
long enough periods of time, a problem encountered by others in serum transfer 
experiments (Inoue et al., 1993). 

In summary, the experiments involving the adoptive transfer of SL splenocytes 
into the BL hosts suggests that amelanosis can be transferred and may be a cell-mediated 
autoimmune process. The role of the associated autoimmune antibodies may not be as 
clear. Whether autoantibodies can cause or trigger the destruction of the melanocytes in 
BL hosts was not apparent. In diabetes, autoimmune antibodies against islet cells is a 
distinguishing feature of the disease, precedes the onset of the disease, are specific to the 
65kDa autoantigen GAD, and can be used as predictive markers of patients susceptible to 
the disease. This likewise may be a similar situation with the autoantibodies in the SL 
chicken. The role of the autoantibodies in diabetes as a primary inducer of diabetes has 
not yet been proven. 






CHAPTER 3 

T CELL RECEPTOR y REPERTOIRE ANALYSIS OF THE EXPANDED 
PERIPHERAL BLOOD y5 T CELL POPULATION DURING AVIAN VITILIGO 

Introduction 

Many autoimmune diseases are T cell-dependent, which, in part, can be 
determined by the study of TCR genes. T cell receptors can generate up to 10 16 total 
receptor specificities by combinatorial and junctional diversity (Janeway and Travers, 
1997), although much of this diversity is lost during thymic education during the 
induction of central tolerance. During an immune response, T cells respond in a clonal 
fashion due to TCR recognition of antigenic peptides. Similarly, during an autoimmune 
response, a fraction of T cells may be clonally expanded in response to self-antigen. The 
T cell response may be described as (1) polyclonal, in which many T cell clones are 
recruited in the response, (2) oligoclonal, in which a small number of T cell clones 
expand, or (3) monoclonal, in which one specific clone responds. Experimentally, this 
can be studied by TCR repertoire analysis of a T cell population. The identification of 
recurrent TCR sequences (with the same or similar CDR3) in large T cell populations 
provides evidence for antigen-driven expansion. 

T cells present in acute graft-versus-host-disease of target organs (skin, liver, and 
intestine) in HLA-matched allogeneic bone marrow transplants express predominantly 



85 



86 

the ap TCR (Dietrich et al., 1994). The TCR Va and Vp usage in both skin and blood 
appeared unrestricted, but with overexpression, unique to each patient, of a few Va and 
Vp gene segments in the skin as compared to blood. To avoid the possibility that a 
nonspecific inflammatory response would mask detection of a clonal T cell expansion, 
Dietrich and colleagues concentrated on one patient. Evidence for the repeated usage of 
at least five specific TCR Vail and Vpi6 transcripts, with CDR3 of unique length and 
sequence, indicated an oligoclonal expansion in the skin, with specific V genes 
overexpressed as compared to the blood, which also showed some of the same recurrent 
TCR transcripts but to a lesser degree. 

Even when genetically identical mice are raised in the same environment, murine 
intestinal intraepithelial lymphocytes (IEL) display unique oligoclonal repertoires. As 
compared to lymph node, Vp expression of aP TCR + IELs is oligoclonal, as indicated by 
prominent and distinct subsets of clonal populations of IELs detected as peaks in their 
CDR3 length analysis (Regnault et al., 1994). This contrasted with the polyclonal 
repertoire of respective T cells in the lymph nodes. 

Likewise, human IELs of five patients were compared (Blumberg et al., 1993). 
The dominant Vp of the IELs in patient 1 was Vp3 and every Vp3 isolated was identical. 
The dominant Vp in patient 2 showed one Vpi3, two Vp5 clones, and an oligoclonal 
expansion of Vp4 and Vp6. Patient 3 displayed either monoclonal or oligoclonal 
dominant Vp gene expression. This was in contrast to a totally polyclonal TCR repertoire 
in the PBL sampling, where no sequence was repeated. Therefore, one or a small number 
of dominant clones appear to comprise the majority of IEL. The conclusions are that 






87 

most IELs are clonally expanded, express a small number of different Vp genes, and may 
recognize a limited number of antigens. Blumberg and colleagues suggest that this small 
T cell repertoire would suffice if the target antigens were conserved, such as bacterial 
heat shock proteins, or a small number of endogenous antigens expressed by IELs in 
response to injury. Similar to the report of Dietrich et al. (1994) on human T cell 
repertoires, the IEL clonal expansion is unique to each individual; there were no shared 
clonal sequences. 

Kourilsky and co-workers applied their high resolution PCR-based method of 
determining and following the TCR repertoire in heterogeneous cell populations of 
tumor-infiltrating lymphocytes (TILs) in human melanomas. Results confirmed clonal 
expansions in a rather complex polyclonal background. Detection of clonal T cell 
expansions before, during, and after treatment is facilitated using this method of PCR- 
detection of junctional diversity in terms of CDR3 length and sequence clonality 
(Puisieux et al., 1994). 

In the Smyth line chicken, the melanocytes, which are located in developing 
feathers and the choroid layer of the eye, appear to be the target of both T and B cell- 
mediated autoimmune responses (Smyth, 1989). Cryosections of the regenerating 
feathers display an intense T cell infiltration including both ap and yd T cell lineages (Erf 
et al., 1995b). In addition, y5 T cells are found in increased proportions in the peripheral 
blood of 40 week-old SL chickens (44.1%) as compared to MHC-matched parental BL 
chickens (33.2%) (Erf et al., 1996) This observation is especially intriguing given the 
fact the chickens have a significantly greater proportion of y5 T cells within the PBL 



88 

compartment than humans and mice, e.g., 20-50% in chicken and 3-5% in human and 
mouse (Bucy et al., 1988). One possible explanation for the expansion of PBL y8 T cells 
is that melanocyte autoantigens caused an antigen-driven clonal expansion in the target 
tissues. The peripheral blood with its higher proportion of y8 T cells, may reflect this 
clonal expansion of y5 T cells infiltrating the feathers in the SL chicken, either as a "spill- 
over" effect from the target tissues. This was observed in the representation of certain 
Vail and Vpi6 TCR transcripts in the blood reflecting the "spillover" of T cells 
infiltrating the skin during human GVHD (Dietrich et al., 1994). 

y5 T cells represent only 0.5-10% of the normal mammalian peripheral blood T 
cell population, and they are believed to be involved in immunity to infectious disease 
(Haas et al., 1993; Modlin et al., 1993; Mombaerts et al., 1993). A role for y8 T cells in 
the pathogenesis of autoimmune disease has been suggested due to their reactivity to 
stress proteins (Kaufman, 1990) and by the accumulation of y8 T cells in affected organs 
and peripheral blood. For example, in systemic sclerosis, y5 T cells expressing the TCR 
VSTgene segment are expanded in both PBL and the lungs, and have restricted 
junctional diversity in terms of CDR3 length and sequence (Yurovsky et al., 1994 and 
1995). This was indicated by a significantly higher proportion of repeated sequences in 
the patients than in controls suggesting that V81 + y5 T cells may be Ag-driven in systemic 
sclerosis patients. The percentage of y8 T cells is expanded in PBL of patients with 
inflammatory bowel disease, showing some skewing of V8 gene expression (Bucht et al., 
1995). Similarly, the frequency of y8 T cells is higher in PBL and cerebrospinal fluid of 
multiple sclerosis patients, as compared to other neurologic disease patients and normal 



individuals, with preferential Vy and V5 gene expression, but heterogeneous clonal 
origins (Stinissen et al., 1995). Systemic lupus erythematosus patients are reported to 
express a diverse PBL Vy repertoire, but an oligoclonal Vy repertoire restricted in terms 
of Vy gene usage and junctional diversity (Olive et al., 1994). Finally, increased levels of 
peripheral blood y8 T cells have been correlated with increased risk for insulin-dependent 
diabetes (Lang et al., 1991). Taken together, these reports suggest a possible role for y5 T 
cells in the development of autoimmune diseases or associated inflammatory processes. 

We tested the hypothesis that the expanded PBL y5 T cell repertoire is involved in 
the pathogenesis of vitiligo in the chicken animal model by characterizing the expressed 
TCR-y repertoire by nucleotide sequence analysis of Vy genes expressed in PBL of 
MHC-matched SL chickens with active vitiligo and control BL chickens. The chicken 
TCR-y locus consists of three families of 8-10 Vy genes, 3 Jy genes, and a single Cy gene 
segment (Six et al., 1997), thus allowing TCR Vy repertoire analysis in the chicken 
animal model. 

Materials and Methods 

Animals 

Breeding colonies were established for the B*®* subline of the SL and BL from 
fertile eggs generously provided by J. Robert Smyth, Jr. (University of Massachusetts at 
Amherst). Chickens were raised at the University of Florida Poultry Science Unit. The 
SL and BL chickens used in this study were MHC-matched and all of the B*0* subline, 
which is characterized by the more severe form of vitiligo in this animal model. The 
degree of pigment loss (amelanosis) was classified according to the following scale after 



90 

Erf et al. (1995b): (1) normal, no apparent amelanosis; (2) mixed amelanosis, with both 
normal and <20% amelanotic feather tissue; (3) mixed amelanosis, with normal and 20- 
60% amelanotic feather tissue; (4) mixed amelanosis, with normal and >60% amelanotic 
feather tissue; and (5) complete amelanosis, all developing feathering tissue is 
amelanotic. 
RT-PCR and cloning 

Peripheral blood lymphocytes were isolated from heparinized blood samples 
collected from the brachial vein by Ficoll-Hypaque density centrifugation. Total RNA 
was prepared using RNeasy columns (Qiagen Corp.), and eluted in RNase-free distilled 
water. Total cDNA was synthesized using random primers and Superscript-II reverse 
transcriptase according to the supplier (Gibco-BRL). 

Rearranged chicken Vy genes were amplified by reverse transcriptase polymerase 
chain reaction (RT-PCR) as previously described (Six et al., 1996) using the following 
Vy family-specific forward primers and a Cy reverse primer: 

Vyl 5*-GCTCTAGACTGAAGCCTGGTTGCATCC-3' 
Vy2 5'-GCTCTAGACCCATACAGAGCCCTGTATCC-3' 
Vy3 5'-GCTCTAGAGCAGACAACATGCTGCTG-3' 
Cy 5'-CCTGGATCCTTTCATAATTCTCTGGTGCTG-3'. 
PCR reactions were performed in 50 ul with 2.5 units Taq polymerase and buffer 
provided with the enzyme (Boehringer Mannheim Biochemicals), cDNA template, 0.2 
mM dNTPs, and 20 pmoles of each primer, for 28 cycles of 60 sec at 94°C, 75 sec at 
55°C, and 90 sec at 72°C. RT-PCR products were cloned into the vector pBluescript-II 



91 

(Stratagene) using restriction sites built into the PCR primers, or cloned into pNoTAT7 
using a Prime PCR Cloner cloning kit (5 Prime 3 Prime, Inc.) by blunt-end ligation. 
Clones were selected by blue/white selection and by size analysis of amplified inserts by 
agarose gel electrophoresis. Individual clones were sequenced using dideoxynucleotide 
cycle-sequencing kits (Applied Biosystems, Incorp.) and T7 and T3 primers. 

Nonincorporated dye terminators were removed on Centri-Sep spin columns 
(Princeton Separations, Adelphia, NJ), and reactions were analyzed on an Applied 
Biosystems Model 373A DNA sequencer. Chromatograms were analyzed using Applied 
Biosystems software. 
DNA sequence comparisons 

For sequence comparisons, only sequences with open reading frames were 
included in the alignments. We have recently described reference sequences for the Vyl, 
Vy2 and Vy3 families, as well as individual Vy family members (Six et al., 1996). The 
ALIGN Plus program (version 2.0, Scientific & Educational Software) was used for 
initial sequence alignments. 

Results 

Phenotvpe of birds used for repertoire analysis 

The pheno types of the SL chickens selected for this study are shown in Table 3-1. 
Table 3-1 . Amelanosis Stage of Smyth Line Chickens at Ages 2-25 Weeks 



Animal # 


2w 


5w 


lOw 


15w 


20w 


25w 


SI 


1 


3 


4 


5 


5 


5 


S2 


1 


1 


3 


4 


4 


4 


S3 


1 


3 


4 


5 


5 


5 


S4 


1 


1 


4 


4 


5 


5 



92 

Two birds (SI and S3) were characterized by early onset of amelanosis, prior to 5 
weeks of age, and three of four SL birds were completely amelanotic at the time of PBL 
collection at 25 weeks of age. As controls, PBL were used from two age-matched BL 
chickens, which had normal plumage. 
TCR Vy5 repertoire analysis 

Rearranged Vyl, Vy2 and Vy3 genes were amplified by RT-PCR from PBL 
mRNA, cloned into a plasmid vector, and individual clones were sequenced. A total of 
86 rearranged Vy genes were sequenced from the PBL of BL and SL chickens. Thirteen 
sequences were eliminated due to the presence of stop codons or frame-shifts in CDR3. 
The presence of these clones was not unexpected, because the PBL were not pre-enriched 
for y8 T cells, and nonproductive TCR-y gene rearrangements would be expected in the 
total PBL population from a0 as well as y8 T cells. The first indication of the 
heterogeneous clonality of the cloned Vy genes from both BL and SL was indicated by 
the fact that only 5 sequences were repeat sequences, i.e. identical to other clones derived 
from the same animal. 

Partial nucleotide sequences for the Vy framework region 3 (FR3), 
complementarity determining region 3 (CDR3) (i.e. the Vy-Jy junction), and FR4 
(encoded by the Jy gene segment), are shown in Figures 3-1, 3-2, and 3-3 for Vyl, Vy2, 
and Vy3 families, respectively. BL and SL Vy sequences are aligned with reference 
sequences we have recently described for Vy gene families in White Leghorn (WL) 
chickens (Six et al., 1996). Identical sequences were obtained from different animals in 
only two cases. Two identical Vy2 sequences were obtained from two different BL 




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99 

animals (clones Vy2-B2.6 and Vy2-B1.2), and identical Vy3 sequences were obtained 
from a BL and SL animal (clones Vy3-B2.3 and Vy3-S2.8). 

Vyl. All eight of the rearranged Vyl genes from BL and three of eleven VI genes 
from SL were identified as known VI family members based on their predicted FR3 
amino acid sequence. The remaining eight of eleven Vyl genes from SL may represent a 
novel Vyl family member, which shares two amino acid substitutions (Gly85Arg and 
Lys89Glu) with three other Vyl genes. Considering the relatively low number of amino 
acid substitutions compared to WL Vyl genes and the absence of further genomic 
mapping and sequence data for the chicken TCR-y locus, these novel SL Vyl genes may 
also represent allelic differences between known Vyl family members segregating in the 
WL and SL strains. 

Vy2. Ten of twelve BL Vy2 genes and eight of fifteen SL Vy2 genes could be 
identified as known Vy2a subfamily members. The remaining two of twelve BL Vy2 
sequences represent a novel Vy2 subfamily, which we designate Vy2d, and the remaining 
seven of fifteen SL sequences represent three different novel Vy2a genes, which may 
represent new Vy2a subfamily members or allelic polymorphisms between WL and SL. 

Vy3. Ten of eleven BL and eight of eleven SL Vy3 genes could be identified as 
known Vy3 family members. The remaining one of eleven BL and three of eleven SL 
sequences represent two different novel Vy3 genes, which may represent new Vy3 family 
members or allelic polymorphisms between WL and SL. There are four SL Vy3 
sequences representing a Vy3 member not seen in BL sequences, suggesting a possible 
shift in Vy3 usage in SL as compared to BL. 



100 

CDR3 length and amino acid composition 

The amino acid sequences encoded by the BL and SL Vy genes are shown in 
Figure 3-4 for the region between the conserved Cys94 and Phel08 residues in Vy and Jy, 
respectively. Sequences were compared for overall CDR3 length, measured as the 
number of amino acids between these two anchor positions, the number of nongermline- 
encoded amino acids in the N region, and for the amino acid content of CDR3 and N 
regions. No differences were found between BL and SL for the CDR3 or N region length 
of all Vy sequences combined or for individual Vy families. When the SL Vy amino acid 
sequences in the CDR3 were compared to BL, there were fewer nonpolar and more 
charged amino acids in Vyl sequences, and more nonpolar and fewer polar amino acids 
in Vy3 sequences. However, none of the differences in amino acid content (frequency of 
nonpolar, polar, and charged amino acids between BL and SL Vy sequences were 
statistically significant as determined by % 2 analysis (data not shown). 
Jy usage 

Overall the usage of the three Jy gene segments in the BL and SL sequences was 
58% vs. 51% usage of Jyl, 13% vs. 19% of Jy2, and 30% vs. 29% of Jy3. Although there 
appeared to be more Vyl -Jyl and Vy3-Jy2 and fewer Vyl-Jy3 and Vy3-Jyl combinations 
in SL chickens as compared to BL chickens, the differences in Jy usage overall and 
within each Vy family are not statistically significant by % 2 analysis (data not shown). 

Discussion 

The function of y5 T cells in general is less well understood than that of ap T 
cells, and they are characterized by functional differences, such as antigen recognition 



101 



Vyl 



VyI-186 

VY1-B2.17 

VY1-B2.24 

Vyl-B2.20 

VY1-B2.15 

VY1-B2.21 

VY1-B2.18 

VY1-B2.2 

VY1-B2.22 

vyi-sa.i 

Vy1-S2 
VY1-S2 
VY1-S2 

Vyl-Sl 
Vyl-81 

VYl-Sl 
VY1-S6 
VyI-SI.7 
Vyl -81. 2 
VYl-S2.ll 



v y 



N region 



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R. 



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W YSAWI . Y . 

PD GDE . I . 

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S 

LR 

LRPGS 

VYG E.I. 

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W 

GGT 

DR 

RAR 

DR 

VN GDE . I . 

V 

SLG 



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VY2-B2.12 

Vy2-B2.6 

VY2-B1.2 

vya-Bi.7 

VY2-B1.6 

VY2-B1.5 

VY2-B2.13 

Vy2-B1.4 

VY2-B1.21 

VY2-B1.9 

VY2-B1.18 

Vy2-Sl.l 

VY2-S6.1 

VY2-S2.13 

VY2-S1.3 

VY2-S1.4 

Vy2-S2.1 

VY2-S2.9 

VY2-S1.8 

VY2-S2 

VY2-SS 

VY2-S1 

Vy2-S1 

VY2-S1 

V Y 2-S1 

Vy2-S6 



V Y 



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10 
4 
9 
2 

6 

14 
7 



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SA 

SA 

SS DE.I. 

VLFD 

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Y AWI . Y . 
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SA 

T 

SA 

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R 

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MS AWI.Y. 

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S WI.Y. 

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VY3-B2.1 RSAWI Y. 

VY3-B2.5 NYR AWI.Y. 

Vy3-B2 .8 .... R G 

Vy3-B2.3 Q H DE.I. 

VY3-B2.2 YQG WSE 

VY3-B2.7 YQG R 

VY3-B1.14 YQ LT WI.Y. 

VY3-B1.16 YQG IYGD 

VY3-B1.13 EV 

Vy3-B2.4 EQGFPT G 

Vy3-S6.2 Q D YSAWI. Y. 

VY3-S1.13 QG LR 

VY3-SS.3 QG SPSY GDE . I . 

VY3-S2.8 Q H DE.I. 

VY3-S2.10 AR 

VY3-S6.1 NPLE AWI.Y. 

V Y 3-S3.3 EP WI.Y. 

VY3-S3 .5 RQ AYI 

VY3-S3.6 RQ AR 

VY3-S6.4 RQ E.I. 

VY3-S2.12 RQ ALSD DE.I. 









Figure 3-4. Predicted amino acid sequences in one letter code of rearranged TCR-y genes from Brown line and Smyth 
line chickens, from the conserved Cys94 and Phel08 residues in Vy and Jy, respectively. Dots indicate identity to the 
top line of sequence. 



102 

and repertoire selection, and usually express an activated phenotype (Haas et al., 1993; 
Sciammas et al., 1991). The presence of y8 T cells in the populations of lymphocytes 
infiltrating tissues affected in various autoimmune diseases suggested a possible role for 
this T cell subset in the pathogenesis of autoimmunity, which has been further supported 
by evidence of clonal expansion of the infiltrating y8 T cells in an antigen-driven fashion 
by findings of related or identical TCR junction sequences (Yurovsky et al., 1994; 
Yurovsky, 1995; Wucherpfennig et al., 1992; Olive et al., 1992; Shimonkevitz et al., 
1993). However, the appearance of y5 T cells after a(5 T cells in some autoimmune 
diseases, such as multiple sclerosis, suggests that they may be recruited secondarily to 
contribute to the inflammatory response (Zhang et al., 1992). The peripheral blood y6 T 
cell pool is also expanded in autoimmune diseases, often in a clonal fashion, such as 
systemic sclerosis (Yurovsky et al., 1994; Yurovsky, 1995), inflammatory bowel disease 
Bucht et al., 1995), multiple sclerosis (Stinissen et al., 1995), systemic lupus 
erythematosus (Olive et al., 1994), and insulin-dependent diabetes (Lang et al., 1991). It 
is unclear whether the expanded peripheral blood y5 T cells are activated in the periphery 
and then migrate into the affected tissue, or represent spill-over from the inflammatory 
response in the affected tissue into the periphery. Increased numbers of peripheral blood 
y8 T cells might also arise from nonspecific polyclonal expansion due to the 
inflammatory process. 

In the avian model of vitiligo, expansion of the peripheral blood y8 T cell 
population is detectable at 13-18 weeks of age, several weeks after onset of disease at 6-8 
weeks, suggesting that their appearance is a secondary event. Human vitiligo patients 



103 

with the nonsegmental form of vitiligo have been studied for changes in their peripheral 
blood lymphocyte subsets by FACS analysis using a panel of mAbs recognizing T cell 
surface markers (Abdel-Nasser et al., 1992). No significant differences were observed in 
T cells positive for ap TCR, y5 TCR, CD3, CD4, CD8, CD45RO, CDllb, CDllc, 
CD16, CD56, CD25, or CD54. The only changes that were detected included a decrease 
in the CD45RA + subset and an increase in HLA-DR + cells, suggesting an increase in 
activated peripheral T cells. Whether this observation is unique to this subset of human 
vitiligo patients or represents a difference in the pathogenesis or progression of vitiligo in 
the SL chicken animal model is unknown. 

In this report we addressed the issue of clonality of the peripheral blood y8 T cells 
in the avian model of vitiligo, and found no evidence for clonal expansion of yd T cells 
belonging to any of the three subgroups of y5 T cells that can be identified based on Vy 
family gene expression. This conclusion is based on analysis of CDR3 length, CDR3 
amino acid content and Vy-Jy gene combinations expressed in SL peripheral blood as 
compared to the control parental BL chicken. It should be noted that the number of y8 T 
cells in the affected tissue, i.e. growing feathers, does increase ten-fold in SL chickens, 
however, due to the even greater expansion of aP T cells, the proportion of y5 T cells in 
the infiltrating T cell population actually is lower than in the feather pulp of normal 
animals (Erf et al., 1995b). It is unknown whether the infiltrating y5 T cells are clonally 
expanded. 

Future studies might include the use of spectratyping of CDR3 lengths to 
determine which Vp and Vy subfamilies show recurrent usage based on the size of the 



104 

CDR3 peaks (Dietrich et al., 1994). This experimental approach might also indicate 
whether there is clonality within the complex polyclonal repertoire. Single strand 
conformational polymorphism (SSCP) analysis of amplified PCR products on a 
nondenaturing polyacrylamide gel would also afford extra characterization of the 
predominance of certain clones. Diffuse bands and smearing would be indicative of 
polyclonality; monoclonality or oligoclonality would be confirmed by one or several 
prominent bands correlating to bulk sequencing. 

The developing feather is the tissue of choice to examine the amelanotic process 
at the site of T cell infiltration. We anticipate that expression of a more restricted T cell 
repertoire might be found in the regenerating feather, compared with the repertoire found 
in the T cells of the peripheral blood lymphocytes of the SL chicken. This has been 
reported for the T cell usage in other autoimmune diseases as well as transplant rejection 
and tumor infiltrations. For example, an oligoclonal TCR repertoire consisting primarily 
of VP8.2 and Voc2 or Va4 has been reported for the mouse (Acha et al., 1988; Urban et 
al., 1988) and rats (Burns et al., 1989) models of EAE. Comparison of the TCR 
transcripts of T cells found in patient bronchiolar lavage to the same patient's PBL 
revealed oligoclonal preferences for V51, Va and V(i gene families which persisted over 
time and from multiple tissues (Yurovsky and White, 1995). 

In conclusion, the expanded y8 T cell population of SL chickens appears to 
represent a polyclonal expansion, with no apparent restriction in junctional diversity or 
significant changes in CDR3 length or amino acid content, or shifts in Jy gene utilization. 
These results suggest that the changes observed in PBL y8 T cells in SL chickens are a 



105 

consequence of the disease process rather than a causal factor, and may be a secondary 
result of the inflammatory response at the sites of melanocyte destruction. 






CHAPTER 4 

ENDOGENOUS VIRAL LOCI IN THE SMYTH LINE CHICKEN: A MODEL FOR 
THE AUTOIMMUNE DISEASE VITILIGO 

Introduction 

Viruses have been implicated in the pathogenesis of many autoimmune diseases. 
Infections by human cytomegalovirus (HCMV) have shown a specific and highly 
significant association with systemic lupus erythematosus, which has also been associated 
with retroviruses and Epstein Barr Virus infections (Rider et al., 1997). After an 
encephalomyelitis infection that has been enhanced by a cryolesion in Lewis rats, 
cervical lymph nodes appear to be a source of autoimmune lymphocytes involved in 
cerebral EAE. Reduction in severity of EAE by lymphadenectomy suggested that the 
lymph nodes prime T cells to target the infected brain (Phillips et al., 1997). There are 
several murine models of virus-induced diabetes, including lymphocytic choriomeningitis 
(LCMV), Coxsackie virus, herpes virus, and encephalomyocarditis virus, which is related 
to the mouse mammary tumor virus (MMTV) and encodes a MHC class Il-dependent 
superantigen via its N-terminal moiety in its env gene (Conrad et al., 1997; Ramsingh et 
al., 1997). 

Several mechanisms have been postulated to explain how viral involvement leads 
to autoimmunity (Aichele et al., 1996; Nakagawa and Harrison, 1996; Barnaba, 1996). 



106 



107 

Viruses are involved in the generation of new epitopes (neoantigens) causing a loss of 
tolerance (breaking of immune ignorance). Immune responses to viral antigenic 
determinants may trigger cross-reactive autoimmune reactions to shared determinants of 
the self-antigens that have been released due to tissue destruction during a host antiviral 
immune response (molecular mimicry) (Douvas and Sobelman, 1991). Viruses may act 
as superantigens that activate T cells expressing specific Vp family genes, for example 
the Mis locus in mice, or they may provide immunosuppression, such as in HIV. 
Respiratory syncytial virus induces host interferon production to inhibit a proliferative 
response by human PBMCs to the infection (Preston et al., 1995). 

In persistent infections in which the body can not completely remove the virus, 
such as in autoimmune hepatitis, the infected tissue is destroyed during long-term chronic 
inflammatory responses to the replicating virus. Thus, the destruction is really due to the 
persistent cytotoxic T cell response on the target tissue. This destruction inadvertently 
and continuously releases large quantities of the organ's self-antigens (especially never 
exposed intracellular antigens) which become presented by professional APCs in 
lymphoid tissue (Koziel et al., 1992; Cerny et al., 1994). Subsequently, self-reactive B 
cells and T cells become activated to secrete autoantibodies and maintain the autoimmune 
response. 

During infection, some viral protein products can modulate or counteract host 
antiviral immune defenses (reviewed by Gooding, 1992; Marrack and Kappler, 1994). 
Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), have often been 
the targets for such viral immune modulation. Shope fibroma virus, a pox virus, produces 



108 

a protein that binds to TNF preventing the recognition of TNF, by its receptors and thus 
preventing the activation of inflammatory responses to remove this virus. Cowpox virus 
encodes a soluble glycoprotein that has amino acid homology to that of the IL-1 receptor. 
This product probably competes with cell-bound IL-1 receptors for secreted IL-1, 
interfering with the activation of IL-1 cytokine-mediated inflammatory responses. The 
product of the BCRF1 gene of the Epstein-Barr virus stimulates the conversion of T cells 
into Th2 helper cells by its structural and functional analogy to IL-10. EBV therefore 
avoids induction of inflammatory responses controlled by Thl cell activity. 

Viruses produce proteins that may subvert the host in other ways as well. Human 
adenoviruses produce virus-associated (VA) RNAs. These transcripts can regulate 
interferon activation of PI kinase by preventing the phosphorylation of eIF-2 (eucaryotic 
protein synthesis initiation factor), which would prevent the synthesis of viral proteins 
(Matthews and Shenk, 1991). VA RNAs therefore facilitate continued growth of the 
virus in the host cells. IgG antibody-dependent complement-mediated destruction of 
virus- containing cells is avoided by herpes viruses. The herpes simplex virus induces the 
infected cell to express HSV-Fc receptor, a heterodimer of glycoproteins E and I that 
binds to the Fc region of the host's nonspecific nonimmune IgG. This binding prevents 
complement-mediated lysis of infected cells by blocking access to the cell surface of 
antiviral antibody or effector cells (Bell et al., 1990). Antibodies to herpes simplex virus 
and CMV Fc-binding proteins have been detected in patients with rheumatoid arthritis 
(Tsuchiya et al., 1993; Williams et al., 1992). In rheumatoid arthritis, the HLA-DR.pi 
alleles contain the QKRAA amino acid sequence in the peptide-binding region that has 



109 

been associated with the autoimmune condition. Enhanced humoral and cellular 
responses to QKRAA sequences expressed by Epstein-Barr virus have been found in RA 
patients (La-Cava et al., 1997). 

Endogenous retroviruses (ev) are germline elements that encode components of a 
retrovirus, are present in all cells, and are inherited in a Mendelian fashion (reviewed in 
Rovigatti and Astrin, 1983). Endogenous retroviruses are passed from parent to offspring 
as stable proviral elements integrated into the germline. By reverse transcription from a 
viral-encoded RNA-dependent DNA polymerase, the DNA copies of these RNA-based 
viruses have become integrated into the genome of the host flanked by a 5' and a 3' long 
terminal repeat (LTR). Three genes vital for viral replication are included: gag, a 
precursor polyprotein; pol, a precursor for protease, reverse transcriptase, and 
endonuclease; and env, the glycoprotein for the viral envelope. The LTR contains 
multiple cis-acting elements involved in gene expression and acts as a promoter. 

Many ev loci are not transcriptionally active; they have truncated open reading 
frames because of termination codons, deletions or frameshift mutations. However, some 
ev loci are expressed and may actually produce viral particles. Their structural and 
sequence similarity to infectious exogenous retroviruses, which have been associated 
with immune dysfunction and tissue-specific expression, make them candidates for 
pathogenic roles in autoimmunity. Exogenous oncogenic retroviruses also integrate into 
the host genome and, in the process, cause insertional mutagenesis. In addition, they are 
not passed from parent to offspring and contain transforming genes such as v-myc,v-src, 
v-erb, and v-myb, which are not present in the leukosis viruses or endogenous viruses. 






110 

Retroviral integrations may cause immune dysregulation of host cellular gene 
expression. As transient transposable elements, they can activate or inactivate the host 
gene by causing gene rearrangements. Retroviruses can undergo gene duplication (of the 
viral and flanking cellular sequences), capture host genes and move host genetic 
information by means of intracellular replication and integration as a provirus (Hughes et 
al, 1981; Tereba, 1981; Rovigatti and Astrin, 1983). The integration of avian leukosis 
virus into the host's proto-oncogene, c-myc, is an example of insertional activation, in 
this case as a promoter insertion. This brings two coding exons of c-myc under the 
transcriptional control of the 3' LTR and causes higher levels of c-myc expression, and 
the dysregulation of c-myc often leads to B cell lymphomas (Coffin, 1991). Other 
insertional mutations include: (1) enhancer insertions in which the provirus is inserted 
upstream of the natural or cryptic promoter; (2) leader insertions, which allow 
readthrough of several transcripts initiated from the provirus 5 'LTR; (3) terminator 
insertions, which install a poly-A signal truncating the transcript, and causing a build up 
of transcript concentration instead of allowing normal turnover; and (4) insertional 
inactivation, if the insertion is in the coding region and disrupts function of the gene. As 
a result of these insertions, the retrovirus genomes often undergo high rates of 
intragenomic rearrangements such as deletions, point mutations, duplications, and 
inversions near the 5' LTR in order to relieve a block to transcriptional expression from 
the 3' LTR Retroviral proviruses can also act as transactivators to the host genome. 

Endogenous viruses are ubiquitous in vertebrate species, including humans, and 
have been extensively studied in White Leghorn lines of the domestic chicken, Gallus 
gallus, where at least 23 endogenous viral (ev) loci have been characterized by Southern 



Ill 

blot analysis of junction fragments, inheritance patterns, and structural analysis by 
restriction mapping and/or DNA sequencing (Humphries et al., 1984; Ronfort et al., 
1990). Chicken ev loci are structurally related to the avian leukosis and Rous sarcoma 
virus group (ALV and RSV). They may express gs (group specific) or chf (chicken 
factor) antigens, which correspond to the viral gag and env gene products, respectively, or 
may produce intact virus (Rovigatti et al., 1983; Smith, 1986). Apparently these 
endogenous proviruses are not essential; ev-negative chickens were found to be normal 
and fertile (Astrin et al., 1979). 

Recent data suggests that human endogenous viruses may be involved in the 
pathogenesis of a variety of human autoimmune diseases, such as diabetes, systemic 
lupus erythematosus, rheumatoid arthritis, psoriasis, and inflammatory neurologic 
diseases (Uronovitz and Murphy, 1996). Unique ev loci have also been reported in two 
chicken models for autoimmune diseases. Ziemecki and coworkers (Ziemiecki et al., 
1996) described a new locus, designated ev22, in the Obese strain (OS), which is 
characterized by autoimmune thyroiditis, a model for the human organ-specific disease 
Hashimoto's thyroiditis. Cosegregation was observed of ev22 with an OS-specific defect 
in immunoendocrine communication (a deficient corticosterone response after 
intravenous injection of lymphokines), but not with T cell hyperproliferation or 
thyroiditis. Sgonc et al. (1995) reported an association of yet another novel locus, 
designated ev23, in the UCD-200 and UCD-206 chicken lines, which are characterized by 
hereditary systemic scleroderma-like connective tissue disease. Although ev23 may not 
play a causal role in systemic scleroderma, it is suggested to contribute to disease 
susceptibility, that is, by prolonging the response of glucocorticoid increasing factors, 



112 

such as interleukin-1. Thyroid diseases, type I diabetes, and rheumatoid arthritis, all 
diseases with autoimmune components, occur with increased frequency in patients with 
vitiligo (Grimes, 1996). 

In this study we sought to determine whether unique ev loci are correlated with 
vitiligo in the SL chicken model by performing Southern blot analysis on genomic DNA 
from BL and SL chickens. We identified novel ev loci not previously described for 
White Leghorn or other chicken lines, which are segregating in BL and SL chicken lines 
in a large number of different combinations or ev genotypes. Although four ev loci, 
designated ev-SLl through ev-SL4, were observed to be present in significantly higher 
proportions in SL than in BL chickens, none of the ev-SL loci was found to be 
exclusively associated with the vitiligo phenotype when ev genotypes for affected and 
nonaffected SL chickens were compared. However, the genetic polymorphisms detected 
between BL and SL chickens by these studies suggest that this animal model may be 
useful for further genetic analysis of vitiligo susceptibility. 

Materials and Methods 

Southern blot analysis 

Genomic DNA samples were prepared from red blood cells of the BL and SL 
chickens using standard protocols we have previously described (McCormack et al., 
1989). Genomic digests were prepared using the restriction endonucleases BamHl and 
EcoRl (New England Biolabs) according to the manufacturer's guidelines, and restriction 
fragments were separated on 0.8% agarose gels. Southern blots were prepared using 
Hybond N + membranes (Amersham), hybridized at 65°C according to Church and Gilbert 



113 

(1984), and washed with 2x SSC, 0.1% SDS at room temperature, followed by 0.1 x SSC, 
0.1% SDS at 65 °C. The hybridization probe consisted of a 32 P-labeled 315 bp Sacl- 
EcoRI restriction fragment from the plasmid pU5L (the generous gift of Dr. Maureen 
Goodenow), representing the U5 region of the avian leukosis virus LTR (Goodenow and 
Haywood, 1987). This probe hybridizes with both the 5' and 3' LTR of avian leukosis 
virus and endogenous viruses (ev loci). Restriction fragments were scored as present or 
absent. Restriction fragment sizes were calculated based comparison to Hindlll 
restriction fragments of lambda phage DNA. A single copy probe for the T cell receptor 
Cp gene segment (Tjoelker et al., 1990) was used as a control to detect partial digestion. 
Statistical analyses 

The frequencies of individual ev loci, identified as BamHl and EcoRI restriction 
fragments or pairs of fragments, were compared using standard statistical tests, including 
X 2 analysis and Fisher's exact test, to determine whether the differences observed between 
the BL and SL, or between SL progressors and nonprogressors, were statistically 
significant. 

Results 

Phenotypic analysis of SL sample population 

Thirty-five SL and 13 BL chickens were used for the Southern blot analysis of ev 
loci. Twenty-four of the 35 SL chickens displayed a maximum amelanosis stage of 3-5 
according to the amelanosis scale of Erf et al. (1995b) and were classified as progressors 
in the analyses described below. Eleven SL chickens showed no sign of amelanosis 



114 

(stage 1) during their lifetime, and were classified as nonprogressors. The amelanosis 
stages are indicated in Table 4-1. 
Southern blot analysis of BL and SL ev loci 

Two restriction endonucleases were selected for Southern blot analysis of BL and 
SL ev loci, based on published work describing ev loci of other chicken lines. BamHl 
was selected based on its use to identify characteristic junction fragments for the ev loci 
in White Leghorn chickens (Humphries et al., 1984; Rovigatti and Astrin, 1983), and 
EcoRl was selected based on its use to characterize the ev loci of Brown Leghorn 
chickens (Ronfort et al., 1991). Composites of representative autoradiographs obtained 
from Southern blots of BamHl and EcoRl digests of BL and SL genomic DNA 
hybridized with the retroviral LTR probe are shown in Figures 4-1 and 4-2, respectively. 
After hybridization with the LTR probe, all blots were stripped and rehybridized with a 
single copy C(3 gene probe, which revealed that the restriction digests were complete 
(data not shown). The frequencies for the presence and absence of ev loci, identified as 
unique BamHl and EcoRl restriction fragment or pairs of fragments as discussed below, 
are summarized in Table 4-2. As discussed below, four ev loci of interest are given the 
designation ev-SL. 

Within the sample population of BL chickens, 11 of 12 birds exhibited different 
restriction digest patterns using BamHl, and 12 of 13 birds exhibited different EcoRl 
patterns. A 12 kb BamHl restriction fragment represent the only ev locus shared by all 
BL chickens. The other most common ev loci in BL chickens are represented by the 7.3 



115 



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kb BamHl restriction fragment (11/12), the 5.8 & 3.4 kb EcoKL fragment pair (12/13), 
and EcoRI restriction fragments of 12.5 kb (1 1/13), 7.2 kb (12/13), and 4.4 kb (12/13). 

Within the SL chicken population sample, 27 of 35 chickens exhibited different 
restriction digest patterns using BamHl, and 26 of 35 birds exhibited different EcoRl 
patterns. Two ev loci appear to be shared by all SL chickens. The first of these is 
represented by the same 7.3 kb BamHl restriction fragment present in 11 of 12 BL 
chickens. The second ev locus present in all SL chickens is represented by the 8.2 kb 
BamHl restriction fragment and by the 1 1 .2 & 1 .3 kb EcoYU fragment pair (designated ev- 
SL2, see below). The other most common ev locus in SL chickens is represented by an 
8.1 kb EcoRl restriction fragment present in 32 of 35 birds (designated ev-SL4, see 
below). 

Five pairs of restriction fragments are present in the same groups of chickens for a 
particular restriction digest. Given the large number of different genotypes segregating in 
the BL and SL populations and the fact that the probe detects both viral LTRs, these 
restriction fragment pairs are likely to represent the 5' and 3' junctions of individual ev 
loci. These restriction fragment pairs include the 9.1 & 3.8 BamHl fragment pairs (ev- 
SL1, Table 4-2), and the 11.2 & 1.3 kb (ev-SL2), 4.2 & 2.8 kb, 8.4 & 3.2 kb, and 5.8 & 
3.4 kb EcoRl fragment pairs (Table 4-2). There were only two exceptions to this pairing 
of fragments, in that one BL bird (BL214) had a 3.2 kb EcoKL band in the absence of a 
8.4 kb EcoKL band, and one SL bird (SL77) had a 3.4 kb EcoKL band in the absence of a 
5.8 kb£coRI band. 












120 

In three instances there is a perfect correlation between the presence of specific 
BamHl and EcoRl fragments or fragment pairs in the same groups of chickens, 
suggesting that they represent the same ev locus (Table 4-2). These include the 9.1 & 3.8 
kb BamHl pair and 4.9 kb EcoRl band, representing the ev-SLl locus, the 8.2 kb BamHl 
band and 1 1 .2 & 1 .3 kb EcoRl pair representing the ev-SL2 locus, and the 1 .4 kb BamHl 
band and 8.4 & 3.2 kb EcoRl pair. 

The number of ev loci per bird for BL and SL chickens is shown in Figure 4-3. 
Ev loci were defined as unique restriction fragment bands or band pairs as described 
above for each restriction enzyme digest (Hughes et al., 1981). The range for the number 
of ev loci per bird was 2-7 for BL and SL chickens, as detected by Southern blots of 
BamHl digests, and 1-9 as detected by Southern blots of EcoRl digests. The average 
number of ev loci per bird did not differ significantly between the BL and SL groups, 
with average numbers of 4.9 ± 1.2 for BL (n=12) and 5.5 ± 1.1 for SL (n=35) chickens 
based on the BamHl Southern blots, and 6.2 ± 1.4 for BL (n=13) and 5.3 ± 1.4 for SL 
(n=35) chickens based on the EcoRl Southern blots. 

The BL and SL BamHl restriction fragments do not co-migrate with the White 
Leghorn loci evl or ev5 (data not shown). The 7.3 kb BamHl fragment present in nearly 
all BL and SL chickens comigrates with ev3 (data not shown), but its identity to ev3 is 
ruled out by the absence of EcoRl fragments of the appropriate size (Hughes et al., 1981). 
Although standard markers for all known ev loci have not been compared to the ev-SL 
loci by Southern blot analysis, comparison of the restriction fragment sizes summarized 
in Table 4-2 with published data for ev loci already reported for White Leghorns 
(Humphries et al., 1984; Rovigatti and Astrin, 1983), Brown Leghorns (Ronfort et al., 





121 








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BL and SL chickens by (A) BamHl and (B) EcoRl restriction digests are plotted against the number of birds with 
each number of loci. Filled bars, BL; open bars, SL. 


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122 

1991) and broilers (Boilliou et al., 1991) suggests that BL and SL chickens are 
characterized by unique ev loci. In particular, none of the restriction fragments 
representing ev-SL loci of interest, i.e. ev-SLl through ev-SL4, correspond in size with 
those of previously identified ev loci. Taken together, these data suggest that a large 
number of previously undescribed ev loci are segregating in BL and SL chicken. 
Comparison of BL and SL ev genotypes 

Statistical analyses of the frequencies for the presence and absence of ev loci in 
BL and SL chickens are shown in Table 4-2 for the BamHl and EcoRI restriction digests. 
Four ev loci, which we have designated as ev-SLl through ev-SL4, were observed to be 
present in significantly higher proportions in SL than in BL chickens (pO.OOl by Fishers 
exact test). The ev-SLl and ev-SL2 loci are identified on both the BamHl and the EcoFJ 
Southern blots, whereas ev-SL3 and ev-SL4 are identified on BamHl or .EcoRI blots, 
respectively (Figures 4-1 and 4-2; Table 4-2). The number of ev-SL loci detected per bird 
and the ev-SL locus combinations observed are shown in Figure 4-4. Most SL chickens 
(32/35) have 3 or more ev-SL loci, whereas most BL chickens (10/12) have one or none. 
As already noted above, ev-SL2 is present in 100% (35/35) of the SL chickens tested, but 
is present in only 38% (5/13) of the BL chickens. The ev-SLl locus is absent from the 
BL sample population, but present in 77% (27/35) of the SL chickens tested. The ev-SL3 
and ev-SL4 loci were present in 74% (26/35) and 91% (32/35) of SL chickens, 
respectively, and in only 17% (2/12) and 8% (1/13) of BL chickens, respectively. As 
shown in Figure 4-4, the only ev-SL combinations observed in either the BL or SL were 
ev-SL2+3, ev-SL 1+2+4, ev-SL2+3+4, and all four ev-SL loci. 



123 



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Other significant differences between BL and SL chickens were observed that 
correlated with the relative absence of some ev loci in SL chickens. As many as seven ev 
loci, which we have not assigned any designations pending further characterization, were 
observed to be present in significantly higher proportions in BL than in SL chickens 
(p<0.01 by Fishers exact test). The ev loci that are present in significantly higher 
proportions of BL than SL chickens include the 12 and 9.5 kb BamHl fragments, and the 
5.8 & 3.4 kb EcoM fragment pair and the 23.5, 12.5, 7.2, and 4.4 kb EcoKI fragments 
(Table 4-2). These data suggest that ev loci may be useful as genetic markers to 
distinguish between BL and SL chickens and/or other chicken lines. 
Comparison of SL progressor and SL nonprogressor ev genotypes 

The ev genotypes were compared for SL subpopulations defined on the basis of their 
maximum stage of amelanosis, in order to determine whether there was any correlation 
between the presence of specific ev loci with the vitiligo phenotype. No significant 
differences in the frequency of individual ev locus presence could be associated with the 
SL progressor (stages 2-5) phenotype, as compared to the SL nonprogressor (stage 1) 
phenotype (Table 4-3). The number of ev-SL loci detected per bird and the ev-SL locus 
combinations present were similar in SL progressors and nonprogressors. These data 
suggest that, in contrast to two other chicken models of autoimmunity (Sgonc et al., 
1995; Ziemiecki et al., 1988), there is no unique ev locus associated with vitiligo in the 
SL chicken. 








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Discussion 

The inheritance of vitiligo in the Smyth line animal model is polygenic in nature, 
and shows low penetrance in out-cross matings (Smyth et al., 1981). The incidence and 
severity of the amelanosis and other associated autoimmune defects (hypothyroidism and 
alopecia-like feathering defect) in the SL chicken are influenced by a variety of factors, 
both genetic and environmental. Although the major histocompatibility complex (MHC) 
is an important genetic factor, as evidenced by differences in disease severity and age of 
onset associated with different MHC alleles in SL sublines (Erf et al., 1995a), the BL and 
SL chickens used in this study were MHC-matched, thus ruling out MHC influence. 
Other genetic factors appear to include inherent melanocyte defects, melanin 
pigmentation genes, melanocyte-stimulating hormone, and sex hormones (reviewed in 
Smyth, 1989). The varying incidence of amelanosis in different SL breeding colonies 
from 60-90% suggests that some environmental factors may be associated with housing 
conditions (McCormack and Smyth, unpublished). 

Human vitiligo also appears to be polygenic, and is suggested to result from 
recessive alleles at several unlinked autosomal loci based on extensive familial 
aggregation of vitiligo (Bhatia et al., 1992; Majumder et al., 1993), and the observation 
that 20% of probands are reported to have at least one first-degree relative affected by 
vitiligo (Nath et al., 1994). It has been speculated that simultaneous alterations in several 
genes are required to cause disease or increase susceptibility, e.g. mutations in genes 
controlling melanocyte growth and survival and/or the immune response (Lacour and 



127 

Ortonne, 1995). Candidate genes for vitiligo susceptibility might be suggested by defects 
in enzymes involved in melanogenesis and catecholamine metabolism that have been 
associated with vitiligo (Halaban and Moellmann, 1993; Austin and Boissy, 1995; Salzer 
and Schallreuter, 1995; Schallreuter et al., 1994). Although there are some reports that 
specific HLA class I or II alleles are associated with vitiligo in some human populations 
(Dunston and Hadler, 1990; Vennecker et al., 1992; Ando et al., 1993; al Fouzan et al., 
1995), other investigators report no such association (Schallreuter et al., 1993; Huang et 
al., 1996). 

In this study we have taken advantage of the genetics of endogenous viruses in 
chickens to explore the possibility that endogenous viruses may play a role in genetic 
susceptibility to vitiligo in the avian model. Nearly all chickens have ev loci, which may 
be silent or expressed as viral proteins or infectious virus, and ev gene expression can 
influence the course of infection by exogenous avian leukosis virus (Rovigatti and Astrin, 
1983). There are reported associations between various specific ev loci and commercially 
important production traits in chickens (Govora et al., 1991; Iraqi et al., 1994), however, 
it was the two reports of association of novel ev loci with two other autoimmune diseases 
that prompted us to examine ev loci in SL chickens, including the OS chicken with 
spontaneous hereditary thyroiditis (Ziemiecki et al., 1988) and the UCD-200 and 206 
lines with hereditary systemic scleroderma-like connective tissue disease (Sgonc et al., 
1995). 

There are several possible roles of ev loci in susceptibility to autoimmune 
diseases. One possibility is that one or more ev loci disrupts a gene expressed in the 
target tissue, contributing to an increased susceptibility to autoimmune recognition, or in 



128 

lymphocytes, resulting in a possible breakdown of normal self-tolerance. An example of 
such an insertional mutagenesis event in an autoimmune disease was reported by Wu and 
colleagues (1993), who observed that integration of an endogenous retrovirus has 
occurred in the Fas apoptosis-regulating gene of MRL-lpr/lpr mice. Alternatively, ev loci 
may represent genetic markers for linked susceptibility genes. A third possibility is that 
one or more ev loci produces infectious viral particles, resulting in a somatic reinfection 
of cells involved in the autoimmune process as suggested above. Whether human 
endogenous viruses are involved in vitiligo remains to be determined, although their 
possible involvement in human neoplastic and autoimmune disease has been suggested 
(Hohenadl et al., 1996; Urnovitz and Murphy, 1996). 

We identified four ev loci that are present at statistically significantly higher 
frequencies in SL than BL chickens, and an additional seven ev loci characteristic of BL 
chickens. The predominance of the ev-SL loci may have originated during the original 
selection of SL chickens and/or may have been introduced during derivation of the SL 
from outcrosses with other chicken lines (Smyth et al., 1981). When the ev genotypes for 
SL progressors and nonprogressors were compared, there was no significant association 
of ev loci with the autoimmune phenotype, thus, in contrast to the OS and UCD-200 
avian models of other human autoimmune diseases, we find no evidence for a role of ev 
loci in the pathogenesis of vitiligo in the SL chicken animal model. 

Possible roles for endogenous viruses in the pathogenesis of avian amelanosis 
might be detectable with additional experiments. DNA methylation has been suggested 
as being partially responsible for the low expression of some avian endogenous viruses. 
An inhibitor of DNA methylation, 5-azacytidine can induce the expression of viral 



129 

particles in ev-\ containing chicken embryo cells (Rovigatti and Astrin, 1983). 5- 
azacytidine treatment activates silent genes and promotes cellular differentiation. 5- 
azacytidine has been shown to increase the incidence of autoimmune thyroiditis in the 
susceptible parental line (Cornell C strain) of the Obese strain chicken (Schauenstein et 
al., 1991). Chronic low dose administration of 5-azacytidine induced amelanosis in the 
genetically susceptible Brown Line chickens, but not the more distantly related LBL 
chickens (Sreekumar et al., 1996). However, the effects of 5-azacytidine treatment on ev 
gene expression in BL and SL chickens have not been reported. 

If experimental evidence is found to implicate a particular ev locus in the 
pathogenesis of vitiligo, it could be identified in a genomic library using appropriate 
probes and restriction mapping, cloned and then sequenced in order to determine 
homology with that of known retroviruses. In situ hybridization of the specific ev 
sequence can be used as a probe to metaphase chromosomes to localize the ev loci. Six 
ev loci in chickens have be mapped to chromosome 1 alone (Tereba et al., 1979; Tereba 
and Astrin, 1980). In addition, the phenotype of the ev locus would be characterized for 
the presence of the gs antigen (indirect immunofiourescence), of the chf (fusion with the 
16Q cell line), detection of the virus (which subgroup) or viral proteins from the culture 
supernatant, and the susceptibility to other subgroups of the ALV-RSV group 
(Humphries et al., 1984a). Endogenous retroviruses are in subgroup E and are 
nononcogenic, unlike the ALVs of the exogenous viruses found in subgroups A, B, C, 
and D. Expression of subgroup E viral proteins helps make the cells refractory to 
infection by other viruses of the same subgroup. Cells containing ev3, ev6, and/or ev9 



130 

have a reduced susceptibility to infection to exogenous subgroup E virus (Rovigatti and 
Astrin, 1983; Robinson et al., 1981). 

The heterogeneity of the endogenous viral loci detected in this study for the SL 
and BL chickens is interesting considering the fact that the SL was derived from the BL. 
This may be due to the outcrossings used to establish the SL (Smyth et al., 1981) i.e., the 
outcrossing to Barred Plymouth Rock, Light Brown Leghorns, and a random-breeding 
meat stock may have introduced additional ev loci. 

An alternative approach to genetic mapping of vitiligo susceptibility is genome- 
wide genetic linkage analysis. Sets of primer pairs for microsatellite genetic markers are 
now available from the U.S. Poultry Gene Mapping Project. Mapping backcross progeny 
with these markers will assist in the identification of candidate regions associated with 
vitiligo. Candidate genes may ultimately be identified within these genomic intervals, 
which can then be analyzed for gene expression, identity of the protein, mutational 
analysis and functional assays to determine the cause of amelanosis. 

One approach to examine gene expression would make use of 5-azacytidine 
treated BL chickens. Northern blot analysis could be used to compare gene expression in 
SL, BL, 5-azacytidine-treated BL, and LBL chickens. One might predict differences in 
expression of candidate genes when comparing the 5-azacytidine-treated BL and SL to 
untreated BL and LBL chickens. 

Interestingly, of the approximately 100 BL chickens we have raised, we have 
recently identified one BL chicken expressing amelanosis (stage 4). The ev genotype of 
this amelanotic BL chicken resembled a SL genotype, i.e. all four ev-SL loci were present 
and several ev loci characteristic of BL chickens were absent (data not shown), 



131 

suggesting that the genome of this individual BL chicken is more similar to SL chickens 
than to other BL chickens. This observation is consistent with the hypothesis that the 1- 
2% incidence of amelanosis observed in BL chickens (Smyth et al., 1981) is due to the 
chance combinations of multiple recessive vitiligo susceptibility genes segregating in the 
Brown line, and that these genes were selected for during the derivation of the Smyth 
line. In conclusion, although we find no evidence of linkage of ev loci with the vitiligo 
phenotype in SL chickens, our results suggest that BL and SL chickens bear considerable 
genetic polymorphisms, and will therefore provide a useful model for further genetic 
dissection of vitiligo susceptibility. 






CHAPTER 5 
SUMMARY AND FUTURE DIRECTIONS 

As autoimmune diseases are explored many have witnessed that finding a simple 
answer is doubtful. The process of amelanosis found in the Smyth line chicken is another 
example of a complex situation. There is obvious evidence that it is a polygenic 
phenomenon. Three sublines of the Smyth line chicken exist as the B 101 , B 102 , and the 
Bl03. The B 101 subline has the quickest onset and greatest severity but all three had a 
high incidence of amelanosis between 80% and 90%. Yet even in the B 101 , there is 
variability. Females usually display a quicker onset, pass through the stages more rapidly, 
and achieve the higher stages of amelanosis than the males. However, not all females 
will develop amelanosis. Some will never become amelanotic, while others do not even 
begin depigmentation until the 17 th week of life and do not progress to the severe stages. 
In the SL2 population, three females reverted and became partially repigmented after they 
became sexually mature. Most of the male B*®' never became amelanotic. Of the male 
B^Ol chickens that did become amelanotic a few became just as severe as the females 
with similar ages in terms of onset and progression. Some and only female SL chickens 
were blind, as noticed by their unawareness when they were being picked up. There were 
females that displayed alopecia, the feathering defect. 

Thus the variability of amelanosis in the Smyth line B subline characterizes the 
polygenic variability in the rate, penetrance and environment. Smyth reported that in 



132 



133 

Massachusetts, 90% of the SL developed amelanosis. In comparison, the UF SL colony 
is characterized by a 60% incidence of amelanosis while the colony of Dr. G. Erf 
(University of Arkansas), which is raised under specific pathogen-free conditions, has an 
even lower incidence of amelanosis (personal communication). The Brown line chickens 
of the same MHC haplotype, B 101 , have a susceptibility of 1-2%. One BL, BL7-1130, 
did spontaneously become amelanotic at the same early onset and severity of condition as 
the typical SL we've studied. The BL hosts in the adoptive transfer experiments that 
demonstrated amelanosis might indeed have had the genetic combination to be more 
susceptible to develop amelanosis on their own, as demonstrated in the BL7-1130. 
Perhaps the transferred cells helped to potentiate and quicken what was already there. 
This would reflect the work of Wicker's lab. They felt that the transfer of splenocytes 
from affected NOD mice into young NOD females yielded the quicker onset and severity 
to already susceptible genetics within the hosts (Wicker et al., 1986). 

The fact that the splenocytes were able to transfer susceptibility to the BL hosts 
is an indication that T cells mediate some aspect of amelanosis. As cited before, the 
works of Austin et al. (1992, 1995), Boissy et al. (1985) and Lamont et al. (1981, 1982) 
have proven a role of autoantibodies by bursectomy, which basically eliminates the B cell 
population in chickens. The anti-melanocyte autoantibodies detected melanocyte proteins 
of 65-80kDa molecular weights, and the latest evidence shows that these represent 
binding to the possible autoantigen Trp-1. The drug cyclosporin, an inhibitor of all T 
cells has been shown to suppress amelanosis in the SL chicken (Pardue, 1987). The 
experiments described herein, provide the first in vivo evidence that T cells can at least 



134 

transfer a potentiation of the amelanotic disease susceptibility found in the Smyth 

chicken. 

It would thus be worth expanding these T cell transfer experiments. As stated 
earlier, fewer restrictions to the experiment need to be in place to better assure its success. 
I would suggest an experiment patterned using a series of repeated donations of affected 
SL splenocytes, using cyclophosphamide to provide the immunosuppression. 
Cyclophosphamide does not affect T cells and untreated chicks are reported to have a 
better survival rate (Toivanen et al., 1975; Lehtonen et al., 1990). A dose dependence 
curve should be generated to optimize the quality of immunosuppression despite the 
sacrifice of an initial set of birds. A more pathogen-limited environment in which to raise 
the immunocompromised hosts during the initial recovery from immunosuppression is 
suggested. The use of 5-azacytidine may help to potentiate the manifestation of the 
amelanotic phenotype of the hosts as seen in the work by Sreekumar and colleagues 
(1995); but it might disguise the cell transfer. Mitogen-stimulation such as by Con-A of 
the donor splenocytes may also help to potentiate the observation of amelanosis in the 
transfer hosts. 

If repeatable results continue, then T cell subsets may further help define the 
involved autoimmune T cells. Separation of chicken T cell subsets is made possible with 
available reagents that include y§ T cells, aP T cells expressing Vpi genes, and aP T 
cells expressing Vp2 genes, which are identified by the mouse monoclonal antibodies, 
TCR1, TCR2, and TCR3, respectively. 



135 

A TCR repertoire analysis of the regenerating feather may characterize the 
expansion that appears to be indicated in the intense lymphocytic infiltration of the pulp 
as reported in the work of Erf et al. (1995a, 1995b). Counts of cells in cryosections 
stained with mouse anti-TCR antibodies have indicated high presence for both CD4 + and 
CD8 + T cells expressing predominantly a(3 TCR bearing Vpi (i.e. TCR2 + ). So the 
repertoire analysis should still be initially examining Vpi. If an oligoclonal expansion of 
certain recurring subset(s) of T cells is detected from the feather analysis, then it might be 
possible to isolate a subset of T cells from regenerating feathers, which could be cultured, 
stimulated with Con-A, and then transferred into 5-6 week BL hosts. This population 
might be more reflective of the autoantigen-activated cells than splenic cells. 

More importantly, this would lead to possible immunotherapy. Monoclonal 
antibodies directed against the specific Vp chain (for example) might be used to 
inactivate that autoreactive subset of T cells while leaving the rest of the T cell repertoire 
intact. This V(3 selective therapy has been performed experimentally in the treatment of 
chronic relapsing EAE in SJL/J mice by Whitham and colleagues (1996). This would 
bridge the work between the TCR repertoire analysis and the adoptive transfer studies. 

By detecting endogenous virus integrations as inheritable stable elements in the 
genomes of the Smyth line chicken, four novel loci (ev-SL) have been identified that may 
be used as genetic markers for vitiligo susceptibility in this chicken. Although these loci 
were not unique to only the SL birds displaying the amelanotic phenotype, it may still be 
possible to find loci that are correlated with the disease. Associations of novel ev-loci 
have been found in the two other major chicken models for autoimmunity, the OS 



136 

chicken for thyroiditis and the UDC-200 and UCD-206 chickens for systemic 

scleroderma. 

An improvement in possibly detecting vitiligo susceptibility genes is to use a 
genome-wide linkage analysis, mapping backcross progeny between the SL and BL 
chickens. Using primer pairs for microsatellite genetic markers from the U.S. Poultry 
Gene Mapping Project, I would like to identify candidate genetic intervals that are 
associated with vitiligo. This would assist in the identification of vitiligo-associated 
candidate regions. The candidate genes may be identified from these candidate regions by 
physical mapping. Yac constructs would be created and linked into contigs. Within each 
Yac construct, a more dense use of microsatellite markers would be used to further 
narrow and fine map the intervals within a candidate region. A candidate gene could be 
identified in a genomic library and then be confirmed by in situ hybridization to 
chromosome spreads using the sequence of a cloned restriction fragment from the 
candidate locus. 

Once a gene is cloned, then the more interesting questions can be examined. I will 
need to translate its protein sequence and look for its identity by a search for homology 
to known proteins in GenBank or similar database. If the protein is well characterized, 
such as IL-4, then functional assays of IL-4 and the pathogenesis of vitiligo could be 
examined. Mutational analysis by introducing a transgene containing a reporter gene 
such as the lac gene or a gentimycin resistance gene could be introduced in the chick 
embryo to create a knockout and see if vitiligo is recreated in a BL. The effects of over- 
or under-expression of the protein can be assessed. 












137 

What has still not been addressed is the inherently defective melanocyte itself. 
Still available are original plans to perform antibody dependent cell cytotoxicity assays. 
These would consist of incubating cultured SL or BL melanocytes (all of the B 101 
haplotype) from cultures with either affected or nonaffected SL sera or BL sera and either 
White Leghorn complement or White Leghorn spleen cells as effectors of cytotoxicity. 
This will examine the surface expression of the SL melanocyte and detect aberrant 
expression of melanocyte autoantigens as compared to that expressed by BL. Such 
autoantigens might be those involved in melanin production, including TRP-1. If the sera 
of the SL can activate the destruction of both BL and SL melanocytes or if the sera of the 
BL can not activate the destruction of either of the melanocytes, then the melanocyte is 
not different between the two strains, at least at the level of surface expression of key 
autoantigens. 

Experiments detecting differences in RNA and protein expression between SL and 
BL melanocytes and T lymphocytes are another direction. This would require that a 
library of primers for chicken cytokines be readily available, unless one is willing to 
optimize the conditions for using mammalian primers. Cultures would include T cells, 
melanocytes and APCs. PCR amplification from mRNA isolated from the cultures would 
allow the detection of the types of cytokines released by the T cells. The identification 
of IL receptors may be detected as well. 

A cDNA library of the mRNA produced by cultured melanocytes could be 
established. From the survey conducted, identity of candidate vitiligo susceptible loci 
can be determined. The cDNA would be cloned into vectors and the DNA of candidate 
vitiligo susceptible loci sequenced. The sequence could be translated into an amino acid 



138 

sequence and compared with sequences submitted in GenBank or similar database. This 
may characterize and reveal the possible aberrant nature of the SL melanocyte. 

The nature of the proteins expressed may reveal possible epitope differences 
between the SL and BL melanocytes. It may confirm or corroborate with the work of 
Boissy and colleagues in the identification of autoantigens (Austin et al., 1992; Austin 
and Boissy, 1995). It may lead to oral immunotherapy experiments using peptide 
administrations similar to the administrations of MBP into EAE mice and GAD into 
NOD mice to induce tolerance. 

Thus, this was my study of amelanosis in the Smyth line chicken as an animal 
model for the autoimmune disease of human vitiligo. Vitiligo is just one piece in the 
autoimmune puzzle. As one of many autoimmune diseases, it is my hope, as well as 
others, that a common factor or theme of factors can be found and that I would in some 
way have helped improve the lives of many. 


















LIST OF REFERENCES 



Abbas, A.K., Lichtman, A.H., and Pober, J.S. 1991. Cellular and Molecular 
Immunology. W.B. Saunders Company. 

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BIOGRAPHICAL SKETCH 

Edmund Chuan-son Leung was born in East Orange, New Jersey, the oldest child 
of George and Ying Leung, a mechanical engineer and a private duty nurse, respectively. 
Ed attended Hofstra University, Hempstead, New York, and graduated in 1982 with a 
B.A. in biology, with a minor in biochemistry. He was employed at the Uniformed 
Services University of the Health Professions, in Bethesda, Maryland, and at Georgetown 
University, Washington, District of Columbia, and was encouraged to apply to graduate 
school. He entered graduate school at the University of Florida, Department of Pathology 
and Laboratory Medicine, in August 1991. After completion of his degree, he will 
continue his academic career at the University of Florida in the laboratory of Dr. Jin 
Xiong She. 






159 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 

Wayne T. McCormack, Ph.D., Chairman 
Associate Professor of Pathology, 

Immunology and Laboratory Medicine 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Mark A. AtkinsonTrh.D. 
Associate Professor of Pathology, 

Immunology and Laboratory Medicine 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 



ireen M. Gpodenow, 



Maureen M. Gj/odenow, Pli.D. 
Associate Professor of Pathology, 

Immunology and Laboratory Medicine 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy 




^Thomas C. Rowe, Ph. 
Associate Professor of Pharmacology 
and Therapeutics 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 

Edward K. Wakeland, Ph.D. 
Professor of Pathology, Immunology and 
Laboratory Medicine 

This dissertation was submitted to the Graduate Faculty of the College of 
Medicine and to the Graduate School and was accepted as partial fulfillment of the 
requirements for the degree of Doctor of Philosophy. 



May, 1998 

Dean, College of Medicine 



$Uj.WfilL-/~/C£ 



Dean, Graduate School 















UNIVERSITY OF FLORIDA 



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